Desmoglein 2 (DSG2) binding proteins and uses therefor

ABSTRACT

The present invention provides recombinant adenoviral compositions and methods for their use in treating disorders associated with epithelial tissues.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/206,448 filed Nov. 30, 2018, issued as U.S. Pat. No. 10,611,803 onApr. 7, 2020, which is a continuation of U.S. patent application Ser.No. 15/593,578 filed May 12, 2017, issued as U.S. Pat. No. 10,150,798 onDec. 11, 2018, which is a continuation of U.S. patent application Ser.No. 14/429,803 filed Mar. 20, 2015, issued as U.S. Pat. No. 9,688,727 onJun. 27, 2017, which is a U.S. national phase filing off of PCTApplication Serial No. PCT/US14/57139 filed Sep. 24, 2014, which claimspriority to PCT Patent Application Serial No. PCT/US13/61431 filed Sep.24, 2013, and to U.S. Provisional Patent Application Ser. No. 61/954,822filed Mar. 18, 2014, each of which is incorporated by reference in itsentirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with U.S. Government support under R01 CA080192and R01 HLA078836 awarded by the National Institutes of Health. The U.S.Government has certain rights in this invention.

BACKGROUND

Human adenoviruses (Ads) have been classified into six species (A to F),currently containing 51 serotypes. Most Ad serotypes utilize thecoxsackie-adenovirus receptor (CAR) as a primary attachment receptor(Bergelson et al., 1997). This is, however, not the case for species BAd serotypes. Recently, we have suggested a new grouping of species BAds based on their receptor usage (Tuve et al., 2006). Group 1 (Ad16,21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3,Ad7, 14) share a common, unidentified receptor/s, which is not CD46 andwhich was tentatively named receptor X; Group 3 (Ad11) preferentiallyinteracts with CD46, but also utilizes receptor X if CD46 is blocked.

Species B Ads are common human pathogens. Since 2005, a simultaneousemergence of diverse species B serotypes at the majority of US militarytraining facilities was observed. This included serotypes Ad3, Ad7, andAd14 (Metzgar et al., 2007). In 2007 a new, highly pathogenic strain andpossibly more virulent strain of Ad14, Ad14a, has been discovered atseveral sites in the US and in Asia (Louie et al., 2008; Tate et al.,2009). We recently demonstrated that Ad14a belongs to species B group 2Ads with regards to their receptor usage (Wang et al., 2009).Collectively, all receptor X utilizing serotypes (Ad3, Ad7, Ad14, Ad14a,and Ad11) are referred to herein as AdB-2/3.

AdB-2/3 have great relevance as gene transfer vectors, particularly withregard to tumors of epithelial origin, representing most solid tumors(Yamamoto and Curiel, 2010). Epithelial cells maintain severalintercellular junctions and an apical-basal polarity. Key features ofepithelial cells are conserved in epithelial cancers in situ and incancer cell lines (Turley et al., 2008). Both CAR and CD46 are oftentrapped in tight and adherence junctions of epithelial cancer cells andare not accessible to Ads that use these attachment receptors (Coyne andBergelson, 2005; Strauss et al., 2009). In contrast, AdB-2/3 efficientlyinfect epithelial cancer cells, which is accomplished in part throughinduction of processes that are reminiscent of Epithelial-to-MesenchymalTransition (EMT) (Strauss et al., 2009). Another distinctive feature ofAdB-2/3 is their ability to produce subviral dodecahedral particlesduring their replication, consisting of Ad fiber and penton base (Norrbyet al., 1967). Penton-Dodecahedra (PtDd) cannot assemble fromfull-length penton base protein, but require spontaneous N-terminaltruncation by proteolysis between residues 37 and 38 (Fuschiotti et al.,2006). This cleaved site is conserved in Ad3, Ad7, Ad11, and Ad14 but isnot present in Ad2 and Ad5. In the case of Ad3 the PtDd are formed at amassive excess of 5.5×10⁶ PtDd per infectious virus (Fender et al.,2005), and it has been suggested that PtDd enhance Ad3 infectivity bydisturbing intercellular junctions, thus favoring virus spreading(Walters et al., 2002).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides isolated polypeptidescomprising the amino acid sequence of any one of SEQ ID NOS: 1-11. Inanother aspect, the present invention provides recombinant AdB-2/3 fiberpolypeptides, comprising:

(a) one or more AdB-2/3 fiber polypeptide shaft domains, shaft domainmotifs, or functional equivalents thereof;

(b) an AdB-2/3 fiber polypeptide knob domain operatively linked to andlocated C-terminal to the one or more AdB-2/3 fiber polypeptide shaftdomains or shaft domain motifs, wherein the AdB-2/3 fiber polypeptideknob domain comprises the polypeptide of any SEQ ID NOS:1-11; and

(c) one or more non-AdB-2/3-derived dimerization domains operativelylinked to and located N-terminal to the one or more AdB-2/3 fiberpolypeptide shaft domains or shaft domain motifs.

In one embodiment, the AdB-2/3 fiber polypeptide does not include anAdB-2/3 fiber polypeptide tail domain. In another embodiment, each shaftdomain or shaft domain motifs selected from the group consisting of anAd3 fiber polypeptide shaft domain or shaft domain motif, an Ad7 fiberpolypeptide shaft domain or shaft domain motif, an Ad11 fiberpolypeptide shaft domain or shaft domain motif, an Ad 14 fiberpolypeptide shaft domain or shaft domain motif, an Ad14a fiberpolypeptide shaft domain, or shaft domain motif combinations thereof,and functional equivalents thereof. In a further embodiment, each shaftdomain or shaft domain motif comprises the amino acid sequence of anyone of SEQ ID NOS:12-18, SEQ ID NO:43-48, or combinations thereof. Inanother embodiment, the dimerization domain comprises an amino acidsequence selected from the group consisting of SEQ ID NO:24 and SEQ IDNO: 25. In a still further embodiment, the recombinant AdB-2/3 fiberpolypeptide comprises or consists of the amino acid sequence of any oneof SEQ ID NO:28-34. In another embodiment, the AdB-2/3 fiber polypeptideis multimerized, such as dimerized. In a further embodiment, the AdB-2/3fiber polypeptide further comprises one or more compounds conjugated tothe recombinant AdB-2/3 fiber polypeptide, such as therapeutics,diagnostics, and imaging agents.

In a further aspect, the present invention provides isolated nucleicacids encoding the isolated peptide or the recombinant AdB-2/3 fiberpolypeptides of the invention, recombinant expression vectors comprisingthe isolated nucleic acids, and host cells comprising the recombinantexpression vectors.

In another aspect, the present invention provides pharmaceuticalcompositions, comprising

(a) the AdB-2/3 fiber multimer of any embodiment or combination ofembodiments of the invention; and a pharmaceutically acceptable carrier.

In a still further aspect, the present invention provides methods forenhancing therapeutic treatment, or diagnosis of a disorder associatedwith epithelial tissue, and/or imaging epithelial tissues, comprisingadministering to a subject in need thereof:

(a) an amount of one or more therapeutics sufficient to treat thedisorder, diagnostic sufficient to diagnose the disorder, and/or imagingagent sufficient to image the epithelial tissue; and

(b) an amount of an AdB-2/3 fiber multimer or pharmaceutical compositionof any embodiment or combination of embodiments of the invention,sufficient to enhance efficacy of the one or more therapeutics,diagnostics, and/or imaging agents.

Exemplary such disorders associated with epithelial tissue include solidtumors, irritable bowel syndrome, inflammatory bowel disorder, Crohn'sdisease, ulcerative colitis, constipation, gastroesophageal refluxdisease, Barrett's esophagus, chronic obstructive pulmonary disease,asthma, bronchitis, pulmonary emphysema, cystic fibrosis, interstitiallung disease, pneumonia, primary pulmonary hypertension, pulmonaryembolism, pulmonary sarcoidosis, tuberculosis, pancreatitis, pancreaticduct disorders, bile duct obstruction, cholecystitis,choledocholithiasis, brain disorders, psoriasis, dermatitis,glomerulonephritis, hepatitis, diabetes, thyroid disorders, cellulitis,infection, pyelonephritis, and gallstones.

In another aspect, the present invention provides methods for treating adisorder associated with epithelial tissue, comprising administering toa subject in need thereof an amount of an AdB-2/3 fiber multimer orpharmaceutical composition of any embodiment or combination ofembodiments of the invention, sufficient to treat the disorder. Inexemplary embodiments, such a disorder may be a viral infection or asolid tumor.

In a further aspect, the present invention provides methods forimproving delivery of a compound to an epithelial tissue, comprisingcontacting the epithelial tissue with one or more compounds to bedelivered to the epithelial tissue; and an amount of an AdB-2/3 fibermultimer or pharmaceutical composition of any embodiment or combinationof embodiments of the invention, sufficient to enhance delivery of theone or more compounds to the epithelial tissue. In exemplaryembodiments, the one or more compounds may be diagnostic or imagingagents.

In a still further aspect, the present invention provides methods forimproving delivery of a substance to a tissue expressing desmoglein 2(DSG2), comprising contacting the tissue expressing DSG2 with

(a) one or more compound to be delivered to the tissue; and

(b) an amount of an AdB-2/3 fiber multimer or pharmaceutical compositionof any embodiment or combination of embodiments of the invention,sufficient to enhance delivery of the one or more compounds to thetissue.

In another aspect, the present invention provides methods for inducingan epithelial to mesenchymal transition (EMT) in a tissue, comprisingcontacting the epithelial tissue with an amount of an AdB-2/3 fibermultimer or pharmaceutical composition of any embodiment or combinationof embodiments of the invention, sufficient to induce EMT.

In a further aspect, the present invention provides methods foridentifying candidate compounds for one or more of treating a disorderassociated with epithelial tissue, improving delivery of a substance toan epithelial tissue, for improving delivery of a substance tissueexpressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3infection comprising

(a) contacting an AdB-2/3 fiber multimer of any embodiment orcombination of embodiments of the invention, to DSG2 under conditions topromote multimer binding to DSG2, wherein the contacting is carried outin the presence of one or more test compounds; and

(b) identifying positive test compounds that compete with the AdB-2/3fiber multimer for binding to DSG2 compared to control;

wherein the positive test compounds are candidate compounds for one ormore of treating a disorder associated with epithelial tissue, improvingdelivery of a substance to an epithelial tissue, for improving deliveryof a substance tissue expressing DSG2, inducing an EMT in a tissue,and/or treating an AdB-2/3 infection.

DESCRIPTION OF THE FIGURES

FIG. 1A-1F. Residues found to be critically involved in binding to DSG2.FIG. 1A) Shown are the amino acid sequences of the Ad3 (SEQ ID NO: 43)and Ad14p1 (SEQ ID NO: 44) fiber knob. Beta sheets present in the Ad3knob (PDB accession number 1H7Z_A) and Ad14 knob (PDB: 3F0Y_A) areindicated by lines. Black arrows indicate residues within the Ad3 fiberknob which, when mutated individually, ablate or reduce binding to DSG2.Compared to the parental strain Ad14(deWit), Ad14p1 had a deletion oftwo amino acid residues within the FG loop of the fiber protein knob(24) indicated by a triangle. FIG. 1B) Schematic structure of dimericAd3 fiber knob mutants. The fiber knob domain and one shaft motif wasfused through a flexible linker to a homodimerizing K-coil domain (SEQID NO: 25) (41). The proteins are self multimerizing and can be purifiedby His-Ni-NTA affinity chromatography. FIG. 1C-FIG. 1F) Analysis ofbinding of dimeric Ad3 fiber knob mutants to soluble DSG2. FIG. 1C andFIG. 1D) Coomassie staining. 10 μg of purified Ad3 fiber knob (unboiled)were loaded per lane. Trimeric forms of the fiber knobs are indicated byan arrow. The gel contained SDS and the loading buffer containing DTT,which caused the disassembly of multimers of trimeric fiber knobs aspreviously reported (41). FIG. 1E and FIG. 1F): Western blot usingsoluble recombinant DSG2 as a probe, followed by anti-DSG2-mAb andanti-mouse IgG-HRP. For comparison, JO-1 (0.5 μg/lane) is shown. TheWestern blots were scanned and signals were quantified.

FIG. 2A-2B. 3D model of the Ad3 fiber knob. The structure is based onPDB accession number 1H7Z_A. FIG. 2A) Four critical areas involved inDSG2 binding. The critical residues are shown on the isosurface of thetrimeric fiber knob. View from the top (apical side) facing thereceptor. FIG. 2B) All critical residues combined. Right side: Anenlargement of the groove after a slight side rotation.

FIG. 3A-3D. Competition of Ad3 virus by dimerized Ad3 knob mutants. FIG.3A) Relative attachment of ³H-labeled Ad3 virus in the presence ofdimeric fiber knob mutants. 1.8×10⁵ HeLa cells were incubated with Ad3knob mutants at a concentration of 2.5 and 100 μg/ml on ice for 1 hour.Then 400 pfu/cell of ³H-Ad3 virus was added on ice for another hour.Unbound virus particles were washed away. Attachment of virus particlesincubated with PBS was taken as 100%. N=3. FIG. 3B) Competition ofAd3-GFP virus infection on HeLa cells. 1.5×10⁵ HeLa cells were seededinto 24 well plates. Cells were incubated with the Ad3 knob mutants atincreasing concentrations for one hour at room temperature. 100 pfu/cellof Ad3-GFP virus were then added and GFP expression was analyzed 18hours later by flow cytometry. FIG. 3B1) percentage of GFP positivecells. FIG. 3B2) mean fluorescence intensity. N=3. The standarddeviation was less than 10%. FIG. 3C) Relative attachment of ³H-labeledAd3 virus in the presence of dimeric fiber knob mutants with multiplemutations. The study was performed as described in FIG. 3B) The standarddeviation was less than 10%. FIG. 3D) Competition of Ad3-GFP virusinfection on HeLa cells. The study was performed as described in FIG.3C) The standard deviation was less than 10%.

FIG. 4A-4B. Analysis of Ad3 fiber knob binding to soluble CD46. Ad3fiber knobs containing different numbers shaft motifs and the wild-typeAd3 fiber knob (lane 1: Ad3-S6/Kn, lane 2: Ad3-S5/Kn, lane 3: Ad3-S4/Kn,lane 4: Ad3-S3/Kn, lane 5: Ad3-S2/Kn, lane 6: Ad3-S/Kn), JO-1 (lane 7)and the CD46-binding Ad35 fiber knob (lane 8) were blotted andhybridized with soluble DSG2 (FIG. 4A) or soluble CD46 (FIG. 4B).Binding was detected by anti-DSG2 mAb or anti-CD46 mAb.

FIG. 5A-5B. Correlation of reduced DSG2 binding with the ability to openepithelial junctions. FIG. 5A) Transepithelial electrical resistance(TEER) measured on polarized colon cancer T84 cells. Cells were culturedin transwell chambers until the TEER was constant, i.e. tight junctionshad formed. A total of 5 μg of dimeric Ad3 fiber knobs in PBS was thenadded for 1 hour to the apical chamber. TEER was measured at theindicated time points. N=6. For time points 1.5 and 4 hours thedifference between JO-1 vs D261N and N186D was significant (p<0.01). Thearrows indicate the addition and removal of Ad3 fiber knobs. FIG. 5B)Enhancement of irinotecan therapy. A total of 4×10⁶ A549 cells wereinjected subcutaneously into CB17-SCID/beige mice. Once the tumorreached a volume of ˜100 mm³ (day 15 after implantation), the mice wereinjected intravenously with 2 mg/kg JO-1, E299V, N186D, or PBS, followedby an intravenous injection of irinotecan (37.5 mg/kg) one hour later.The treatment was repeated on day 25. N=5. The differences between thegroups “irinotecan” vs “E299V+irinotecan” or “irinotecan” vs“N186+irinotecan” were not significant. The difference between“irinotecan” vs “JO-1+irinotecan” was significant (p<0.01) from day 20on.

FIG. 6A-6C. Amino acid substitutions that increase the binding to DSG2.FIG. 6A) Shown is the amino acid sequence of the Ad3 fiber knob (SEQ IDNO: 44). Beta sheets are indicated by lines. Arrows indicate residueswithin the Ad3 fiber knob which, when mutated yielded stronger signalsin colony blot assays, indicating stronger binding to DSG2. FIG. 6B) Theisosurface of the three knob monomers. FIG. 6B1) Top view; FIG. 6B2)Side View. V239 and Y250 are not exposed at the top suggesting astructural change in the knob rather than an involvement in directbinding to DSG2. FIG. 6C) Localization of all mutations that enhance thebinding to DSG2. Residues are show in magenta in two knob monomers.Isosurface of one monomer is shown in grey transparency.

FIG. 7A-7B. SPR analysis of non-dimerized Ad3 fiber knob interactionswith DSG2. FIG. 7A) DSG2 was immobilized on sensorchips, and backgroundwas automatically subtracted from the control flow cell. The Ad3 fiberknobs (w/o dimerization domain: “noDD”) were injected for 3 minutes at2.5 μg/ml followed by a 2.5 minutes dissociation period. FIG. 7B)Summary of SPR data. A concentration range from 2.5 to 10 μg/ml of theknobs has been injected and kinetics and affinity parameters have beenevaluated using the BIAeval software. The extracted data are resumed inthe table. Wt=Ad3 fiber knob without mutations

FIG. 8A-8F. Electron microscopy and 3D structure of Ad3 fiber knobmutant JO-2. FIG. 8A-FIG. 8C) Negative staining of JO-2 with SST.Dimeric forms can be seen but higher organizations are also visible, anheterogeneous complex of around 50 nm depicted by thin arrows and asmaller regular “dodecahedral-like” particle depicted by thick arrows.Close-up views are presented in FIG. 8B and FIG. 8C. FIG. 8D-FIG. 8G)Crystallographic structure of the non-dimerized form of (K217E/F224Smutant). FIG. 8D) protein crystals. FIG. 8E) The wild-type Ad3 knob iscolored in gray with the EF loop 217-224 separately colored. This is theloop which becomes disordered in the mutant. There is no density forthese residues in the mutant structure. FIG. 8F) The mutant is displayedas a cartoon. FIG. 8G) Overlay of these two structures shows that the EGloop is completely disordered in the K217E/F224S mutant. The bottompanels show close-up views of one monomer. K217 and F224 appear assticks.

FIG. 9A-9C. Analysis of dimeric Ad3 fiber knob mutants with increasedaffinity to DSG2. FIG. 9A) Competition of Ad3-GFP virus infection onHeLa cells with dimeric affinity-enhanced mutant Y250F and JO-1 (dimericwt Ad3 fiber knob). The experimental setting is as described for FIG.3C. FIG. 9A1) percentage of GFP positive cells. FIG. 9A2) meanfluorescence intensity. N=3. The standard deviation was less than 10%.FIG. 9B) Competition of Ad3-GFP virus infection on HeLa cells by Ad3knob mutants with enhanced DSG2 binding but without dimerization domain.1.5×10⁵ HeLa cells were seeded into 24 well plates. Cells were incubatedwith the Ad3 knob mutants at the increasing concentrations for one hourat room temperature. 100 pfu/cell of Ad3GFP virus were then added andGFP expression was analyzed 18 hours later. FIG. 9B1) % of GFP+cells;FIG. 9B2) MFI. FIG. 9C) TEER on colon cancer T84 cells. The experimentalsetting was the same as for FIG. 5A. The TEER at 4 hours is shown. N=3.

FIG. 10A-10C. Combination of affinity-enhanced JO-1 versions withchemotherapy. FIG. 10A) Enhancement of irinotecan (I) therapy. Theexperimental setting was the same as in FIG. 5B. The differences in thegroups “JO-1+I” vs “JO-2+irinotecan” and “JO-2+I” vs “JO-4+I weresignificant from day 20 on. N=5. FIG. 10B) JO-4 enhances PLD therapy inan ovarian cancer model at lower dose than JO-1. Mammary fat pad tumorswere established from primary ovarian cancer ovc316 cells. Treatment wasstarted when tumors reached a volume of 100 mm³. Mice were injectedintravenously with 2 mg/kg JO-1 or with 0.5 mg/kg JO-4, followed by anintravenous injection of PEGylated liposomal doxorubicin (PLD) (1 mg/kg)one hour later. Treatment was repeated weekly. FIG. 10C) JO-4 enhancestherapy in poor-prognosis triple negative breast cancer (TNBC). A totalof 4×10⁶ TNBC MDA-MB-231 cells were injected into the mammary fat pad ofCB17 SCID-beige mice. JO-4 (2 mg/kg) was intravenously injected 1 hourbefore the application of cetuximab (C) (10 mg/kg, i.p.) andnab-paclitaxel (nab-P) (5 mg/kg, i.v.). Treatment was given weekly. N=10P<0.01 at day 25 for nab-P+C vs JO-4+nab-P+C.

FIG. 11A-11C. Pharmacokinetics, toxicity, and immunogenicity of JO-4.FIG. 11A) Serum clearance of JO-1 and JO-4. hDSG2 transgenic mice withsubcutaneous TC1-hDSG2 tumors (˜600 mm³) were intravenously injectedwith JO-1 or JO-4 (2 mg/kg) and serum samples were analyzed for byELISA. N=3. Note that the y-axis has a log scale. FIG. 11B1) Lymphocyteand FIG. 11B2) platelet counts in hDSG2/TC1-hDSG2 transgenic mice afterJO-1 or JO-4 injection. N=3. FIG. 11C) Therapy studies inimmunocompetent hDSG2 transgenic mice with TC1-hDSG2 tumors. When tumorsreached a volume of ˜80 mm³, JO-1 or JO-4 (2 mg/kg) or PBS was injectedintravenously followed one hour later by PLD/Doxil (i.v. 1.5 mg/kg).Treatment was repeated as indicated by arrows. Tumors were then allowedto re-grow for about two weeks. From day 15 on serum anti-JO-1/J)-4antibodies were detectable by ELISA. Two more treatment cycles wereperformed at day 28 and day 35. JO-1 and JO-4 continued to be effectiveafter multiple treatment cycles, even in the presence of detectableantibodies. The difference between JO-1/PLD vs JO-4/PLD is significantfrom day 31 on. N=10.

FIG. 12. Alignment of fiber knob sequences. The residues thatablate/reduce Ad3 knob binding to DSG2 are indicated (from top to bottomSEQ ID NOs: 14, 15, 16, 17).

FIG. 13. Sera from humans and hypervaccinated mice do not inhibitactivity of JO-4. Analysis of human serum for binding with JO-4 byELISA. Rabbit polyclonal antibodies against the Ad3 fiber knob were usedfor capture, followed by recombinant JO-1 protein, human serum(dilutions 1:20 to 1:1000), and anti-human IgG-HRP. Commercial human Abserum depleted for IgG was used as a negative control (horizontal line).Serum from a scientist who routinely works with Ad3 virus was used as apositive control. P1 to P38 are serum sample for ovarian cancer patientsobtained from the Pacific Ovarian Cancer Research Consortium.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine(Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q),glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

As used herein, the abbreviation “Ad” refers to an adenovirus and istypically followed by a number indicating the serotype of theadenovirus. For example, “Ad3” refers to adenovirus serotype 3.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides isolated polypeptidecomprising or consisting of the amino acid sequence:

(SEQ ID NO: 1) TLWTG(V/P)(N/K)P(----/T)(E/R)ANC(Q/I)(M/I)(M/E)(Y/A/N/D)(S/G)(S/K)(E/Q)(S/N)(N/P)D(C/S)KL(I/T)L(I/T)LVK(T/N)G(A/G)(L/I)V(T/N)(A/G)(F/Y)V(Y/T)(V/L)(I/M)G(V/A)S(N/D)(N/D/Y)(F/V)N(M/T)L(T/F)(T/K)(Y/H/N)(R/K)N(I/V)(N/S)(F/I)(T/N)(A/V)EL(F/Y)FD(S/A)(A/T)G(N/H)(L/I)L(T/P)(S/R/D)(L/S)SSLKT(P/D)L(N/E)  X2   X3 (S/Y)(G/K)Q(N/T)(M/--)(A/--)(T/--)(G/--)A(I/L/D)  X4 (N/S)A(K/R)(S/G)FMPSTTAYPF  X5 (--/L)(N/P)(N/D/V)(N/A)(S/G)(R/T)(E/H)(N/K/--)  X6  N  X7  I(Y/F)G(T/Q)C(H/Y)Y X8 ASD(H/G/R)(T/A)(A/L)FP(I/L)(D/E)(I/V)(S/T)VMLN(Q/R/K)R(A/L)(I/L/P)(R/N/D)(A/D/N/S)(D/E/R)TSY(C/V)(I/M)(R/T)(I/V/F)(T/L)WS(W/L)  X9  (T/A)G(D/L/V)APE(G/V/--)(Q/--)T(S/T)(A/Q)(T/A)TL(V/I)TSPFTF(Y/S)YIR EDD;wherein

X2 is H, L, or P;

X3 is K or E;

X4 is T, F, S, or L;

X5 is V, D, or is absent;

X6 is E, G), or is absent

X7 is Y or F;

X8 is T, K, or E; and

X9 is N or S;

wherein at least one of the following is true:

X2 is P;

X3 is E;

X4 is S, or L;

X5 is D;

X6 is G);

X7 is F;

X8 is E; or

X9 is S.

Isolated polypeptides according to this aspect of the invention comprisemutant AdB-2/3 knob domains, which can be used, for example, to producerecombinant AdB-2/3 fiber polypeptides that provide significantlyenhanced affinity for desmoglein 2 (DSG2) compared to previously knownDSG2 binding polypeptides. As shown in the examples that follow,recombinant AdB-2/3 fiber polypeptides of the invention that incorporatethe mutant knob domains of this first aspect of the invention arefurther shown to be therapeutically more potent than previously knownDSG2 binding polypeptides for treating epithelial-associated disorder,exemplified by improved efficacy in a series of cancer models. Theisolated peptides of the invention can also be used, for example, asantigens against AdB-2/3 viruses.

In one embodiment, the isolated polypeptides of the first aspect of theinvention comprises or consists of the amino acid sequence

(SEQ ID NO: 2) TLWTG(V/P)(N/K)P(E/R)ANC(Q/I)(M/I)(M/E)(Y/A/N/D)(S/G)(S/K)(E/Q)(S/N)(N/P)D(C/S)KL(I/T)L(I/T)LVK(T/N)G(A/G)(L/I)V(T/N)(A/G)(F/Y)V(Y/T)(V/L)(I/M)G(V/A)S(N/D)(N/D/Y)(F/V)N(M/T)L(T/F)(T/K)(Y/H/N)(R/K)N(I/V)(N/S)(F/I)(T/N)(A/V)EL(F/Y)FD(S/A)(A/T)G(N/H)(L/I)L(T/P)(S/R/D)(L/S)SSLKT(P/D)L(N/E)  X2   X3 (S/Y)(G/ K)Q(N/T)A(I/L/D) X4  (N/S)A(K/R)(S/G)FMPSTTAYPF  X5(--/L)(N/P)(N/D/V)(N/A)(S/G)(R/T)(E/H)(N/K/--)  X6 N  X7 I(Y/F)G(T/Q)C(H/Y)Y  X8  ASD(H/G/R)(T/A)(A/L)FP(I/L)(D/E)(I/V)(S/T)VMLN(Q/R/K)R(A/L)(I/L/P)(R/N/D)(A/D/N/S)(D/E/R)TSY(C/V)(I/M)(R/T)(I/V/F)(T/L)WS (W/L)  X9 (T/A)G(D/L/V)APET(S/T)(A/Q)(T/A)TL(V/I)TSP FTF(Y/S)YIREDD.

In another embodiment, the isolated polypeptides of the first aspect ofthe invention comprises or consists of the amino acid sequence

(SEQ ID NO: 3) TLWTG(V/P)(N/K)P(E/R)ANC(Q/I)(M/I)(M/E)(Y/A/N/D)(S/G)(S/K)(E/Q)(S/N)(N/P)D(C/S)KL(I/T)L(I/T)LVK(T/N)G(A/G)(L/I)V(T/N)(A/G)(F/Y)V(Y/T)(V/L)(I/M)G(V/A)S(N/D)(N/D/Y)(F/V)N(M/T)L(T/F)(T/K)(Y/H/N)(R/K)N(I/V)(N/S)(F/I)(T/N)(A/V)EL(F/Y)FD(S/A)(A/T)G(N/H)(L/I)L(T/P)(S/R/D)(L/S)SSLKT(P/D)L(N/E)  X2   X3 (S/Y)(G/ K)Q(N/T)A(I/L/D) X4  (N/S)A(K/R)(S/G)FMPSTTAYPF  X5  L(N/P)(N/D/V)(N/A)(S/G)(R/T)(E/H)(N/K/)  X6  N  X7  I(Y/ F)G(T/Q)C(H/Y)Y X8  ASD(H/G/R)(T/A)(A/L)FP(I/L)(D/E)(I/V)(S/T)VMLN(Q/R/K)R(A/L)(I/L/P)(R/N/D)(A/D/N/S)(D/E/R)TSY(C/V)(I/M)(R/T)(I/V/F)(T/L)WS(W/L)  X9  (T/A)G(D/L/V)APET(S/T)(A/Q)(T/A)TL(V/I)TSPFTF(Y/S)YIRE DD.

In a further embodiment, the isolated polypeptides of the first aspectof the invention comprises or consists of the amino acid sequence

(SEQ ID NO: 4) TLWTGPKPEA NCIIEYGKQN PDSKLTLILV KNGG(I/L)VNGYVTLMGASDYVN TLFKNKNVSI NVELYFDATG HILPDSSSLK TDLE X2 X3 YKQT AD  X4 SARGFMP STTAYPF X5 LP NAGTHN X6 N X7  I FGQCYY  X8 ASD GALFPLEVTV MLNKRLPDSR TSYVMTFLWS L X9 AGLAPETT QATLITSPFT FSYIREDD.

In all of these embodiments, at least one of the following is true:

X2 is P;

X3 is E;

X4 is S, or L;

X5 is D;

X6 is G);

X7 is F;

X8 is E; or

X9 is S.

In various embodiments, at least 2, 3, 4, 5, 6, 7, or all 8 of thesestatements is true. In one exemplary embodiment, at least X7 is F. Inanother embodiment, at least X3 is E and X4 is S. In another embodiment,at least X9 is S. In a further embodiment, at least X5 is D. In anotherembodiment, at least X4 is L. In another embodiment, at least X2 is Pand X8 is E. IN another embodiment, at least X6 is G and X8 is E.

In various further embodiments, the isolated polypeptide comprises orconsists of one of the following peptides:

(a) (SEQ ID NO: 5) TLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENFIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (b) (SEQ ID NO: 6)TLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELEYKQTADSSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (c) (SEQ ID NO: 7)TLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLSAGLAPETTQATLITSPFTFSYIREDD; (d) (SEQ ID NO: 8)TLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFDLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (e) (SEQ ID NO: 9)TLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGLVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLEPKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYEASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (f) (SEQ ID NO: 10)TLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADLSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; and (g) (SEQ ID NO: 11)TLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNGNYIFGQCYYEASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD.

In a second aspect, the present invention provides recombinant AdB-2/3fiber polypeptide, comprising:

(a) one or more AdB-2/3 fiber polypeptide shaft domains, shaft domainmotifs or functional equivalents thereof,

(b) an AdB-2/3 fiber polypeptide knob domain, operatively linked to andlocated C-terminal to the one or more AdB-2/3 fiber polypeptide shaftdomains or shaft domain motifs, wherein the AdB-2/3 fiber polypeptideknob domain comprises the polypeptide of any embodiment or combinationof embodiments of the first aspect of the invention; and

(c) one or more non-AdB-2/3-derived dimerization domains operativelylinked to and located N-terminal to the one or more AdB-2/3 fiberpolypeptide shaft domains or shaft domain motifs.

As used herein, “AdB-2/3” is any adenovirus serotype that uses DSG2 asan epithelial 5 cell receptor for viral binding. To date, Ad3, Ad7,Ad11, Ad14, and Ad14a serotypes have been identified. As other Adserotypes are identified, those of skill in the art can readily identifythose that belong to the AbD-2/3 family based on DSG2 binding assays asdisclosed herein. For example, surface plasmon resonance (SPR) studiesusing sensors containing immobilized recombinant DSG2 can be used todetermine if new Ad serotypes bind to DSG2, combined with DSG2competition studies. Further exemplary studies, such as loss and gain offunction analyses, are described in detail in WO 2011/156761.

The adenovirus virion is an icosahedron characterized by a fiber locatedat the base of each of the 12 vertices of the capsid. The fiber on thevirion is a homotrimeric structure consisting of 3 individual fiberpolypeptides. Each adenovirus fiber polypeptide is an asymmetricalstructure consisting of an N-terminal tail, which interacts with thepenton base protein of the capsid and contains the signals necessary fortransport of the protein to the cell nucleus; a shaft, which contains anumber of 15-residue repeating units; and a C-terminal knob domain thatcontains the determinants for receptor binding (J. S. Hong and J. A.Engler, Journal of Virology 70:7071-7078 (1996)). All adenovirusesattach to their receptors through the knob structure on the end of thefiber. Thus, as used herein, the term AdB-2/3 “fiber polypeptide” refersto a full length fiber polypeptide that comprises an N-terminal taildomain, a shaft domain or shaft domain motif, and a C-terminal knobdomain. The fiber polypeptides spontaneously assemble into homotrimers,referred to as “fibers,” which are located on the outside of theadenovirus virion at the base of each of the twelve vertices of thecapsid.

In a preferred embodiment, the recombinant polypeptides do not include atail domain from an Ad fiber polypeptide. As is disclosed in detailbelow, the inventors identified critical residues, mutation of whichresult in fiber polypeptides with significantly enhanced affinity forDSG2, and with significantly enhanced therapeutic potency. Thepolypeptides of this aspect of the invention can thus be used, forexample, to form AdB-2/3 fiber multimers for use in the various methodsof the invention discussed above. In this aspect, the recombinantpolypeptides can include shaft domains or shaft domain motifs from anyAdB-2/3 virus, or any mutants (substitutions, additions, deletions,chimeras, etc.) to such shaft domains or shaft domain motifs that retainor improve binding affinity to DSG2, and are capable of formingmultimers (such as dimers) via the dimerization domain (functionalequivalents). For example, surface plasmon resonance (SPR) studies usingsensors containing immobilized recombinant DSG2 can be used to determineif recombinant polypeptides being assessed bind to DSG2, combined withDSG2 competition studies.

As used throughout the present application, the term “polypeptide” isused in its broadest sense to refer to a sequence of subunit aminoacids. The polypeptides of the invention may comprise L-amino acids,D-amino acids (which are resistant to L-amino acid-specific proteases invivo), or a combination of D- and L-amino acids. The polypeptidesdescribed herein may be chemically synthesized or recombinantlyexpressed. The polypeptides may be linked to other compounds to promotean increased half-life in vivo, such as by PEGylation, HESylation,PASylation, glycosylation, or may be produced as an Fc-fusion or indeimmunized variants. Such linkage can be covalent or non-covalent as isunderstood by those of skill in the art.

As used herein, the term “operatively linked” refers to an arrangementof elements wherein the domains are configured so that they function asa unit for their intended purpose. The term does not require that thedomains are immediately adjacent on the polypeptide, as spacer/linkersequences may be present between the domains, the lengths of which canbe quite variable. In one non-limiting embodiment, the spacer lengthbetween any two domains of the recombinant AdB-2/3 fiber polypeptidescan be between about 0 amino acids and about 20 amino acids. In variousother non-limiting embodiments, the spacer length can be 0-20, 0-19,0-18, 0-17, 0-16, 0-15, 0-14, 0-13, 0-12, 0-11, 0-10, 0-9, 0-8, 0-7,0-6, 0-5, 0-4, 0-3, 0-2, 0-1, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14,1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20,2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8,2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13,3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17,4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20,5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8,5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11,6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13,7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14,8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14,9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14,10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14,11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13,13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18,14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19,16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, 19-20, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acidsin length.

As used herein, “recombinant polypeptide” means a non-naturallyoccurring protein product, wherein the domains of the recombinantpolypeptide are derived from one or more other proteins or artificiallyderived sequences, such as the mutant knob domain polypeptides of theinvention. For example, each shaft domain or shaft domain motif can bederived from a different naturally occurring protein. The recombinantpolypeptide may be constructed by a variety of mechanisms including, butnot limited to, standard DNA manipulation techniques and chemicalassembly via subunit parts of the recombinant polypeptide. The chemicalassembly may lead to an equivalent form as the molecular genetic form oralternative associations with equivalent function. In a preferredembodiment, the recombinant polypeptide is produced by standardrecombinant DNA techniques. Techniques for such recombinant productionand isolation of the recombinant polypeptides of the invention are wellwithin the level of skill in the art based on the teaching herein.

In one embodiment, each shaft domain or shaft domain motifs selectedfrom the group consisting of an Ad3 shaft domain or shaft domain motif,an Ad5 shaft domain motif, an Ad7 shaft domain or shaft domain motif, anAd 11 shaft domain or shaft domain motif, an Ad 14 shaft domain or shaftdomain motif, an Ad14a shaft domain, or shaft domain motif, combinationsthereof, and functional equivalents thereof. The shaft domain or shaftdomain motifs required for fiber knob dimerization, which is requiredfor binding to DSG2 and resulting transient opening of intercellularjunctions. As used herein, a “shaft domain motif” is any portion of ashaft domain that permits fiber knob dimerization of the recombinantAdB-2/3 fiber polypeptides of the invention. Such shaft domain motifscan be readily determined by those of skill in the art, based on theexamples provided below. For example, surface plasmon resonance (SPR)studies using sensors containing immobilized recombinant DSG2 can beused to determine if recombinant polypeptides being assessed bind toDSG2, combined with DSG2 competition studies. Further exemplary studies,such as loss and gain of function analyses, are described in detail inExample 1.

The recombinant polypeptides may comprise between 1 and 22 AdB-2/3 fiberpolypeptide shaft domains or shaft domain motifs. Thus, in variousembodiments to polypeptides comprise 1-22, 1-21, 1-20, 1-19, 1-18, 1-17,1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4,1-3, 1-2, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13,2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-22, 3-21, 3-20,3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8,3-7, 3-6, 3-5, 3-4, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15,4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-22, 5-21, 5-20,5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8,5-7, 5-6, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13,6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17,7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-22, 8-21, 8-20,8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-22,9-21, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10,10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13,10-12, 10-11, 11-22, 11-21, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15,11-14, 11-13, 11-12, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16,12-15, 12-14, 12-13, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16,13-15, 13-14, 14-22, 14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 15-16, 16-22, 16-21, 16-20,16-19, 16-18, 16-17, 17-22, 17-21, 17-20, 17-19, 17-18, 18-22, 18-21,18-20, 18-19, 19-22, 19-21, 19-20, 20-22, 20-21, 21-22, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22AdB-2/3 fiber protein shaft domains or shaft domain motifs. Where morethan 1 AdB-2/3 fiber protein shaft domain or shaft domain motif ispresent, each shaft domain or shaft domain motif can be identical, orone or more copies of the shaft domain or shaft domain motif may differin a single recombinant polypeptide. In a preferred embodiment, therecombinant AdB-2/3 fiber polypeptide has a single shaft domain or shaftdomain motif.

In another embodiment, one or more (or all) shaft domains or shaftdomain motifs in the recombinant polypeptide comprise or consist of anamino acid sequence according to SEQ ID NO 12:

GVL(T/S)LKC(L/V)(T/N)PLTT(T/A)(G/S)GSLQLKVG(G/S)GLTVD(D/T)T(D/N)G(T/F/S)L(Q/K/E)ENI(G/S/K)(A/V)(T/N)TPL(V/T)K(T/S)(G/N)HSI(G/N)L(S/P)(L/I)G(A/P/N)GL(G/Q)(T/I)(D/E)(E/Q)NKLC(T/S/A)KLG(E/Q/N)GLTF(N/D)S(N/S)N(I/S)(C/I)(I/A)(D/N/L)(D/K)N(I/--)NTL;

or SEQ ID NOS:43-48:

Ad3 shaft domain motif: SEQ ID NO: 43 NSIALKNNTL Ad7 shaft domain motif:SEQ ID NO: 44 NSNNICINDNINTL Ad5 shaft domain motif: SEQ ID NO: 45GAITVGNKNNDKLTL Ad11 shaft domain motif: SEQ ID NO: 46 NSNNICIDDNINTLAd14 shaft domain motif: SEQ ID NO: 47 NSNNICIDDNINTLAd35 shaft domain motif: SEQ ID NO: 48 GDICIKDSINTL

In this sequence and other variable sequences shown herein, the variableresidues are noted within parentheses, and a “−” indicates that theresidue may be absent.

In another embodiment, one or more (or all) shaft domains or shaftdomain motifs in the recombinant polypeptide comprise or consist of anamino acid sequence according to SEQ ID NO 13:

GVLTLKCLTPLTTTGGSLQLKVGGGLT(V/I)DDTDG(T/F)L(Q/K)ENI(G/S)ATTPLVKTGHSIGL(S/P)LG(A/P)GLGT(D/N)ENKLC(T/A)KLG(E/Q)GLTFNSNNICI(D/N)DNINTL

or SEQ ID NOS: SEQ ID NOS:43-48

In a still further embodiment, one or more (or all) shaft domains orshaft domain motifs in the recombinant polypeptide comprise or consistof an amino acid sequence selected from the group consisting of SEQ IDNO: 14 (Ad3), SEQ ID NO: 15 (Ad7), SEQ ID NO: 16 (Ad 11), SEQ ID NO: 17(Ad14), SEQ ID NO:18 (Ad14a), and SEQ ID NOS:43-48.

The AdB-2/3 fiber polypeptide knob domain comprises or consists of anyembodiment or combination of embodiments of the first aspect of theinvention (i.e.: any of SEQ ID NOS: 1-11); these polypeptide domains aredescribed in detail in the first aspect of the invention.

As used herein a “dimerization domain” is a peptide sequence thatpromotes dimerization in the recombinant polypeptide that contains it.Any suitable non-AdB-2/3-derived dimerization domain can be used in therecombinant polypeptide of the invention, so long as it permitsdimerization of the recombinant polypeptide and thus binding to DSG2.The dimerization domain is non-AdB-2/3-derived, in that it is not anaturally occurring domain in an AdB-2/3 fiber polypeptide. Non-limitingexamples of the numerous dimerization domains known to those of skill inthe art and suitable for use in the present invention include, but arenot limited to peptide helices containing at least one helix, or astructure formed by a helix, a coil and another helix, etc., coiled coilstructures, dimerization domains within, for example, many cell surfacesignaling receptors, Fc regions or hinge regions of an antibody, leucinezippers, the STAT protein N terminal domain, FK506 binding protein, theLexA protein C-terminal domain, nuclear receptors, the FkpA N-terminaldomain, orange carotenoid protein from A. maxima, M1 matrix protein frominfluenza, neuraminidase from influenza virus, E. coli fuculosealdolase; and the like. (see, e.g., O'Shea, Science. 254: 539 (1991),Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211: 121-128(1996); Klemm et al., Annu. Rev. Immunol. 16: 569-592 (1998); Klemm etal., Annu. Rev. Immunol. 16: 569-592 (1998); Ho et al., Nature. 382:822-826 (1996); and Pomeranz et al., Biochem. 37: 965 (1998)). Furtherexamples include residues 325 to 410 in the bovine papillomavirus E2protein, (Dostatni, N., et al., EMBO J 7 (1988) 3807-3816; Haugen, T.,et al. EMBO J 7 (1988) 4245-4253; McBride, A., et al., EMBO J 7 (1988)533-539; McBride, A., et al., Proc Natl Acad Sci USA 86 (1989) 510-514),Type I deiodinase (D1): DFLVIYIEEAHASDGW (SEQ ID NO: 19) orADFL-YI-EAH-DGW (SEQ ID NO: 20); HIV-1 Capsid Protein:QGPKEPFRDYVDRFYKTLRA (SEQ ID NO: 21); leucine zipper dimerization motifof yeast GCN4: HMKQL D VEEL S NYHL N VARL K VGER (SEQ ID NO: 22);leucine zipper in Escherichia coli transcriptional antiterminatorprotein; and BglG: GVTQLMREMLQLIKFQFSLNYQEESLSYQRLVT (SEQ ID NO: 23). Inpreferred embodiments, the dimerization domain comprises one or morecopies of EVSALEK (SEQ ID NO:24) and/or KVSALKE (SEQ ID NO: 25).

It is well within the level of skill in the art to identify appropriatepeptide sequences that can serve as dimerization domains, and mutantsthereof, in the recombinant polypeptides of the present invention. Forexample, dimerization of the recombinant AdB-2/3 fiber polypeptides canbe assessed by criteria including sedimentation in sucrose gradients,resistance to trypsin proteolysis, and electrophoretic mobility inpolyacrylamide gels (Hong and Engler, Journal of Virology 70:7071-7078(1996)).

The recombinant polypeptides may comprise one or morenon-AdB-2/3-derived dimerization domains. Thus, in various embodiments,the recombinant polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore non-AdB-2/3-derived dimerization domains. Where multiple domainsare present in a polypeptide, it is preferred that each dimerizationdomain is the same.

In a preferred embodiment a spacer peptide is located between thedimerization domain and the one or more shaft domains or shaft domainmotifs. In a further preferred embodiment, the spacer peptide is apeptide with structural flexibility. Virtually any peptide withstructural flexibility can be used. As an example, the flexible peptidemay comprise repetitions of amino acid residues, such as Gly-Gly-Gly-Ser(SEQ ID NO: 26), or any other suitable repetition of amino acidresidues. In another embodiment, the hinge region of an antibody can beused. The spacer can be any suitable length that maintains the abilityof the recombinant polypeptide to dimerize and to maintain binding ofthe recombinant polypeptide to DSG2.

In one preferred embodiment, the recombinant AdB-2/3 polypeptidecomprises one or more shaft domains that each comprise or consist of anAd3 shaft domain (SEQ ID NO:14)

This preferred embodiment can be used with any embodiment or combinationof embodiments described herein. For example, any suitable knob domaincan be used, and any suitable dimerization domain can be used, includingbut not limited to one or more copies of EVSALEK (SEQ ID NO:24) and/orKVSALKE (SEQ ID NO: 25). Similarly any suitable spacer peptides can beused between the dimerization domain and the shaft domain or shaftdomain motif and/or between the shaft domain or shaft domain motif andthe knob domain. In a most preferred embodiment, the recombinant AdB-2/3polypeptide comprises or consists of JO-1 (SEQ ID NO:27), or a multimerthereof (such as a dimer).

The recombinant polypeptides may comprise further domains, such as adomain for isolation of the polypeptide and/or a detection domain. Anisolation domain can be added to facilitate purification/isolation ofthe polypeptide following, for example, recombinant polypeptideproduction. Any suitable isolation domain can be used, including but notlimited to HIS, CBP, CYD (covalent yet dissociable NorpD peptide), StrepII, FLAG, HPC (heavy chain of protein C) peptide tags, GST and MBPaffinity tags. As used herein, “detection domain” means one or moreamino acid sequence that can be detected. Any suitable detection domaincan be used, including but not limited to, inherently fluorescentproteins (e.g. Green Fluorescent Proteins and fluorescent proteins fromnonbioluminescent Anthozoa species), cofactor-requiring fluorescent orluminescent proteins (e.g. phycobiliproteins or luciferases), andepitopes recognizable by specific antibodies or other specific naturalor unnatural binding probes, including, but not limited to, dyes, enzymecofactors and engineered binding molecules, which are fluorescently orluminescently labeled.

In further preferred embodiments, the recombinant AdB-2/3 fiberpolypeptide comprises or consists of the amino acid sequence of one ofthe following

(a) (M/-) (SEQ ID NO: 28)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENFIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (b) (M/-) (SEQ ID NO: 29)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELEYKQTADSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (c) (M/-) (SEQ ID NO: 30)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLSAGLAPETTQATLITSPFTFSYIREDD; (d) (M/-) (SEQ ID NO: 31)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFDLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (e) (M/-) (SEQ ID NO: 32)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGLVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLEPKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYEASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (f) (M/-) (SEQ ID NO: 33)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADLSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; and (g) (M/-)(SEQ ID NO: 34) GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNGNYIFGQCYYEASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD. 

In another embodiment, the recombinant polypeptides are in a multimericform, such as a dimer, trimer, etc. In a preferred embodiment, amultimer comprises a dimer formed by dimerization through thedimerization domains in each homotrimer (ie: a polypeptide is ahomotrimer through trimerization of the knob domain) In multimeric form(such as a dimer), the recombinant polypeptides comprise AdB-2/3 fibermultimers, and can be used in the various methods of the inventiondiscussed above. As will be understood by those of skill in the art,such multimers may comprise multimers of identical recombinantpolypeptide of the invention, or may comprise multimers of differentrecombinant polypeptides of the invention. In one embodiment, thedimerization domains are the same in each recombinant polypeptideforming part of the multimer. In another embodiment, the dimerizationdomains are different in each recombinant polypeptide forming part ofthe multimer. In another embodiment, the shaft and/or knob domains arethe same in each recombinant polypeptide forming part of the multimer.In another embodiment, the shaft and/or knob domains are different ineach recombinant polypeptide forming part of the multimer.

AdB-2/3 fiber multimerization can be determined according to methodswell known to the practitioners in the art. For example, multimerizationof the recombinant AdB-2/3 fiber constructs can be assessed by criteriaincluding sedimentation in sucrose gradients, resistance to trypsinproteolysis, and electrophoretic mobility in polyacrylamide gels (Hongand Engler, Journal of Virology 70:7071-7078 (1996)). Regardingelectrophoretic mobility, the fiber multimer is a very stable complexand will run at a molecular weight consistent with that of a multimerwhen the sample is not boiled prior to SDS-PAGE. Upon boiling, however,the multimeric structure is disrupted and the protein subsequently runsat a size consistent with the protein monomer.

The recombinant polypeptides, or multimeric versions thereof, may bestored in solution or frozen.

In another embodiment, the recombinant polypeptides of the invention arecombined with (such as conjugated to) one or more therapeutics for adisorder associated with epithelial tissue. Such conjugates can be used,for example, in the therapeutic methods of the invention. Methods forconjugating the polypeptides of the invention to a therapeutic ofinterest, such as by covalent binding or chemical cross-linking, arewell known to those of skill in the art. Any suitable therapeutic can beused to form a conjugate according to this embodiment of the invention,including but not limited to tumor stroma degrading compounds (such asrelaxin), alkylating agents, angiogenesis inhibitors, antibodies,antimetabolites, antimitotics, antiproliferatives, aurora kinaseinhibitors, apoptosis promoters (for example, Bcl-xL, Bcl-w and Bfl-1)inhibitors, activators of death receptor pathway, Bcr-Abl kinaseinhibitors, BiTE (Bi-Specific T cell Engager) antibodies, biologicresponse modifiers, cyclin-dependent kinase inhibitors, cell cycleinhibitors, cyclooxygenase-2 inhibitors, growth factor inhibitors, heatshock protein (HSP)-90 inhibitors, demethylating agents, histonedeacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,inhibitors of apoptosis proteins (IAPs) intercalating antibiotics,kinase inhibitors, mammalian target of rapamycin inhibitors, microRNA'smitogen-activated extracellular signal-regulated kinase inhibitors,multivalent binding proteins, non-steroidal anti-inflammatory drugs(NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP)inhibitors, platinum chemotherapeutics, polo-like kinase (Plk)inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs,receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids,small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitorsand the like.

Exemplary therapeutics falling within these various classes include, butare not limited to: docetaxel, doxorubicin, irinotecan, paclitaxel(Taxol®), paclitaxel albumin bound particles (Abraxane®), doxorubicinHCL liposome (Doxil®), BiTE antibodies such as adecatumumab (MicrometMT201), blinatumomab (Micromet MT103) and the like, siRNA-basedtherapeutics, alkylating agents including altretamine, AMD-473, AP-5280,apaziquone, bendamustine, brostallicin, busulfan, carboquone, carmustine(BCNU), chlorambucil, CLORETAZINE.® (laromustine, VNP 40101M),cyclophosphamide, dacarbazine, decitabine, 5′-azacytidine, estramustine,fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU),mafosfamide, melphalan, mitobronitol, mitolactol, nimustine, nitrogenmustard N-oxide, ranimustine, temozolomide, thiotepa, TREANDA®(bendamustine), treosulfan, rofosfamide and the like; angiogenesisinhibitors including endothelial-specific receptor tyrosine kinase(Tie-2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors,insulin growth factor-2 receptor (IGFR-2) inhibitors, matrixmetalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9(MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR)inhibitors, thrombospondin analogs, vascular endothelial growth factorreceptor tyrosine kinase (VEGFR) inhibitors and the like;antimetabolites including ALIMTA® (pemetrexed disodium, LY231514, MTA),5-azacitidine, XELODA® (capecitabine), carmofur, LEUSTAT® (cladribine),clofarabine, cytarabine, cytarabine ocfosfate, cytosine arabinoside,decitabine, deferoxamine, doxifluridine, eflornithine, EICAR(5-ethynyl-1-.beta.-D-ribofuranosylimidazole-4-carboxamide),enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or incombination with leucovorin, GEMZAR® (gemcitabine), hydroxyurea,ALKERAN® (melphalan), mercaptopurine, 6-mercaptopurine riboside,methotrexate, methotrexate analogs (such as trimetrexate andpralatraxate), mycophenolic acid, nelarabine, nolatrexed, ocfosfate,pelitrexol, pentostatin, raltitrexed, Ribavirin, triapine, trimetrexate,S-1, tiazofurin, tegafur, TS-1, vidarabine, and the like; Bcl-2 proteininhibitors including AT-101 ((−)gossypol), GENASENSE® (G3139 oroblimersen (Bcl-2-targeting antisense oligonucleotide)), IPI-194,IPI-565, N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobe-nzenesulfonamide)(ABT-737),N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide(ABT-263), GX-070 (obatoclax) and the like; Bcr-Abl kinase inhibitorsinclude DASATINIB® (BMS-354825), GLEEVEC® (imatinib) and the like; CDKinhibitors including AZD-5438, BMI-1040, BMS-032, BMS-387, CVT-2584,flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, seliciclib(CYC-202, R-roscovitine), ZK-304709 and the like; EGFR inhibitorsincluding ABX-EGF, anti-EGFR immunoliposomes, EGF-vaccine, EMD-7200,ERBITUX® (cetuximab), HR3, IgA antibodies, IRESSA® (gefitinib), TARCEVA®(erlotinib or OSI-774), TP-38, EGFR fusion protein, TYKERB® (lapatinib)and the like; ErbB2 receptor inhibitors include CP-724-714, CI-1033(canertinib), HERCEPTIN® (trastuzumab), TYKERB® (lapatinib), OMNITARG®(2C4, petuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974, EKB-569,PI-166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti-HER/2neubispecific antibody, B7.her2IgG3, AS HER2 trifunctional bispecificantibodies, mAb AR-209, mAb 2B-1 and the like; histone deacetylaseinhibitors include romidepsin, LAQ-824, MS-275, trapoxin,suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid and the like;HSP-90 inhibitors including 17-AAG-nab, 17-AAG, CNF-101, CNF-1010,CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, MYCOGRAB® (humanrecombinant antibody to HSP-90), NCS-683664, PU24FC1, PU-3, radicicol,SNX-2112, STA-9090 VER49009 and the like; activators of death receptorpathways including TRAIL, antibodies or other agents that target TRAILor death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab,ETR2-ST01, GDC0145, (lexatumumab), HGS-1029, LBY-135, PRO-1762 andtrastuzumab; platinum chemotherapeutics include cisplatin, ELOXATIN®(oxaliplatin) eptaplatin, lobaplatin, nedaplatin, PARAPLATIN®(carboplatin), satraplatin, picoplatin and the like; VEGFR inhibitorsincluding AVASTIN® (bevacizumab), ABT-869, AEE-788, axitinib (AG-13736),AZD-2171, CP-547,632, IM-862, MACUGEN (pegaptamib), NEXAVAR® (sorafenib,BAY43-9006), pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584),SUTENT® (sunitinib, SU-11248), VEGF trap, ZACTIMAThi (vandetanib,ZD-6474) and the like; dendritic cell therapy (sipuleucel-T, Provenge®);topoisomerase inhibitors including aclarubicin, 9-aminocamptothecin,amonafide, amsacrine, becatecarin, belotecan, BN-80915, CAMPTOSAR®(irinotecan hydrochloride), camptothecin, dexrazoxine, diflomotecan,edotecarin, ELLENCE® or PHARMORUBICIN® (epirubicin), etoposide,exatecan, abraxane, irenotecan,10-hydroxycamptothecin, gimatecan,lurtotecan, mitoxantrone, orathecin, pirarbucin, pixantrone, rubitecan,sobuzoxane, SN-38, tafluposide, topotecan and the like; antibodiesincluding AVASTIN® (bevacizumab), CD40-specific antibodies, chTNT-1/B,denosumab, ERBITUX® (cetuximab), HUMAX-CD4® (zanolimumab), IGF IR-specific antibodies, lintuzumab, PANOREX® (edrecolomab), RENCAREX® (WXG250), RITUXAN® (rituximab), ticilimumab, trastuzimab and the like;hormonal therapies including ARIMIDEX® (anastrozole), AROMASIN®(exemestane), arzoxifene, CASODEX® (bicalutamide), CETROTIDE®(cetrorelix), degarelix, deslorelin, DESOPAN® (trilostane),dexamethasone, DROGENIL® (flutamide), EVISTA® (raloxifene), AFEMA®(fadrozole), FARESTON® (toremifene), FASLODEX® (fulvestrant), FEMARA®(letrozole), formestane, glucocorticoids, HECTOROL® (doxercalciferol),RENAGEL® (sevelamer carbonate), lasofoxifene, leuprolide acetate,MEGACE® (megesterol), MIFEPREX® (mifepristone), NILANDRON® (nilutamide),NOLVADEX® (tamoxifen citrate), PLENAXIS® (abarelix), prednisone,PROPECIA® (finasteride), rilostane, SUPREFACT® (buserelin), TRELSTAR®(luteinizing hormone releasing hormone (LHRH)), VANTAS® (Histrelinimplant), VETORYL® (trilostane or modrastane), ZOLADEX® (fosrelin,goserelin) and the like; immunologicals including interferon alpha,interferon alpha-2a, interferon alpha-2b, interferon beta, interferongamma-1a, ACTIMMUNE® (interferon gamma-1b) or interferon gamma-nl,combinations thereof and the like. Other agents include ALFAFERONE®(IFN-alpha), BAM-002 (oxidized glutathione), BEROMUN® (tasonermin),BEXXAR® (tositumomab), CAMPATH® (alemtuzumab), CTLA4 (cytotoxiclymphocyte antigen 4), decarbazine, denileukin, epratuzumab, GRANOCYTE®(lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010(anti-CTLA-4), melanoma vaccine, mitumomab, molgramostim, MYLOTARG™(gemtuzumab ozogamicin), NEUPOGEN® (filgrastim), OncoVAC-CL, OVAREX®(oregovomab), pemtumomab (Y-muHMFG1), PROVENGE® (sipuleucel-T),sargaramostim, sizofilan, teceleukin, THERACYS® (BacillusCalmette-Guerin), ubenimex, VIRULIZIN® (immunotherapeutic, LorusPharmaceuticals), Z-100 (Specific Substance of Maruyama (SSM)), WF-10(Tetrachlorodecaoxide (TCDO)), PROLEUKIN® (aldesleukin), ZADAXIN®(thymalfasin), ZENAPAX® (daclizumab), ZEVALIN®. (90Y-Ibritumomabtiuxetan) and the like; ofatumumab; biological response modifiers agentsincluding krestin, lentinan, sizofuran, picibanil PF-3512676 (CpG-8954),ubenimex and the like; pyrimidine analogs include cytarabine (ara C orArabinoside C), cytosine arabinoside, doxifluridine, FLUDARA®(fludarabine), 5-FU (5-fluorouracil), floxuridine, GEMZAR®(gemcitabine), TOMUDEX® (ratitrexed), TROXATYL® (triacetyluridinetroxacitabine) and the like; purine analogs including LANVIS®(thioguanine) and PURI-NETHOL® (mercaptopurine); antimitotic agentsincluding batabulin, epothilone D (KOS-862),N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,ixabepilone (BMS 247550), paclitaxel, TAXOTERE® (docetaxel), PNU100940(109881), patupilone, XRP-9881 (larotaxel), vinflunine, ZK-EPO(synthetic epothilone) and the like; and other chemotherapeutic agentssuch as ABRAXANE® (ABI-007), ABT-100 (farnesyl transferase inhibitor),ADVEXIN® (Ad5CMV-p53 vaccine), ALTOCOR® or MEVACOR® (lovastatin),AMPLIGE®. (poly I:poly C12U, a synthetic RNA), APTOSYN® (exisulind),AREDIA® (pamidronic acid), arglabin, L-asparaginase, atamestane(1-methyl-3,17-dione-androsta-1,4-diene), AVAGE® (tazarotene), AVE-8062(combreastatin derivative) BEC2 (mitumomab), cachectin or cachexin(tumor necrosis factor), canvaxin (vaccine), CEAVAC® (cancer vaccine),CELEUK® (celmoleukin), CEPLENE® (histamine dihydrochloride), CERVARIX®(human papillomavirus vaccine), CHOP® (C: CYTOXAN® (cyclophosphamide);H: ADRIAMYCIN® (hydroxydoxorubicin); O: Vincristine (ONCOVIN®); P:prednisone), CYPAT® (cyproterone acetate), combrestatin A4P, DAB(389)EGF(catalytic and translocation domains of diphtheria toxin fused via aHis-Ala linker to human epidermal growth factor) or TransMID-107R®(diphtheria toxins), dacarbazine, dactinomycin,5,6-dimethylxanthenone-4-acetic acid (DMXAA), eniluracil, EVIZON™(squalamine lactate), DIMERICINE® (T4N5 liposome lotion),discodermolide, DX-8951f (exatecan mesylate), enzastaurin, EPO906(epithilone B), GARDASIL® (quadrivalent human papillomavirus (Types 6,11, 16, 18) recombinant vaccine), GASTRIMMUNE®, GENASENSE®, GMK(ganglioside conjugate vaccine), GVAX® (prostate cancer vaccine),halofuginone, histerelin, hydroxycarbamide, ibandronic acid, IGN-101,IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonasexotoxin, interferon-.alpha., interferon-.gamma., JUNOVAN® or MEPACT®(mifamurtide), lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine(hexadecylphosphocholine), NEOVASTAT® (AE-941), NEUTREXIN® (trimetrexateglucuronate), NIPENT® (pentostatin), ONCONASE® (a ribonuclease enzyme),ONCOPHAGE® (melanoma vaccine treatment), ONCOVAX® (IL-2 Vaccine),ORATHECIN® (rubitecan), OSIDEM® (antibody-based cell drug), OVAREX® MAb(murine monoclonal antibody), paclitaxel, PANDIMEX® (aglycone saponinsfrom ginseng comprising 20(S)protopanaxadiol (aPPD) and20(S)protopanaxatriol (aPPT)), panitumumab, PANVAC®-VF (investigationalcancer vaccine), pegaspargase, PEG Interferon A, phenoxodiol,procarbazine, rebimastat, REMOVAB® (catumaxomab), REVLIMID®(lenalidomide), RSR13 (efaproxiral), SOMATULINE® LA (lanreotide),SORIATANE® (acitretin), staurosporine (Streptomyces staurospores),talabostat (PT 100), TARGRETIN® (bexarotene), TAXOPREXIN®(DHA-paclitaxel), TELCYTA® (canfosfamide, TLK286), temilifene, TEMODAR®(temozolomide), tesmilifene, thalidomide, THERATOPE® (STn-KLH), thymitaq(2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazolinedihydrochloride), TNFERADE® (adenovector: DNA carrier containing thegene for tumor necrosis factor-.alpha.), TRACLEER® or ZAVESCA®(bosentan), tretinoin (Retin-A), tetrandrine, TRISENOX®. (arsenictrioxide), VIRULIZIN®, ukrain (derivative of alkaloids from the greatercelandine plant), vitaxin (anti-alphavbeta3 antibody), XCYTRIN®(motexafin gadolinium), XINLAY® (atrasentan), XYOTAX® (paclitaxelpoliglumex), YONDELIS® (trabectedin), ZD-6126, ZINECARD® (dexrazoxane),ZOMETA® (zolendronic acid), crizotinib, zorubicin and the like.

In another preferred embodiment, the therapeutic comprises a compoundthat binds to desmoglein-2; preferably a compound that binds to DSG2 andopens up tight junctions.

In other embodiments, the therapeutic comprises radioactiveparticles/radiation therapy. Any suitable radioactive therapy orparticle can be used as deemed appropriate by an attending physician,including but not limited to cobalt-60, iodine-131, iridium-192,strontium-89, samarium 153, rhenium-186 and lead-212.

In a preferred embodiment, the therapeutic is an anti-tumor therapeuticand comprises a chemotherapeutic or anti-tumor monoclonal antibody asdescribed herein. In a further preferred embodiment, the anti-tumortherapeutic comprises an antibody selected from the group consisting oftrastuzumab, cetumiximab, petuzumab, apomab, conatumumab, lexatumumab,bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab,edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab,epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomabdaclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab.Non-limiting examples of useful anti-tumor mAb and their specific usesare listed in Table 1, and as further described in Campoli, M., et al.,Principles & Practice of Oncology 23(1&2): 1-19 (2009), incorporatedherein by reference.

TABLE 1 Tumor-Antigen Specific mAbs for Cancer Treatment AntibodyIsotype Target Disease Indication SGN-75 humanized CD70 solid tumors,including IgG1 renal cell cancer, CD70 + hematologic malignanciesTrastuzumab humanized HER2/neu HER2/neu(+) breast IgG1 cancer* CetuximabChimeric IgG1 EGFR EGFR(+) colon cancer* Panitumumab Fully human EGFREGFR(+) colon cancer* IgG2 Matuzumab Humanized EGFR non-squamousnon-small IgG1 cell lung cancer (NSCLC), head and neck squamous cellcarcinoma (HNSCC), breast and pancreatic cancer, colon cancer (CC)Pertuzumab Humanized EGFR NSCLC, HNSCC, CC, IgG1 breast and ovariancancer Ipilimumab Humanized CTLA-4 NSCLC, RCC, (MDX-010) IgG1 metastaticmelanoma Tremelimumab Humanized CTLA-4 NSCLC, RCC, (CP-675, 206) IgG1metastatic melanoma Sibrotuzumab Humanized FAP** NSCLC, CC IgG1DR-4-specific Humanized TRAIL NSCLC, CC, ovarian mapatumumab IgG1cancer, multiple (TRM-1, myeloma, HGS-ETR1) DR-5-specific HumanizedTRAIL solid tumors lexatumumab IgG1 (HGS-ETR2, TRA-8) CantuzumabHumanized CanAg*** CC, pancreatic mertansine IgG1- cancer maytansinoidBevacizumab humanized vascular colon cancer*, (Avastatin) IgG1endothelial non-squamous growth non-small cell lung factor cancer(NSCLC)*, (VEGF) metastatic breast cancer*

In another embodiment, the recombinant polypeptides of the invention arecombined with (such as conjugated to) one or more diagnostic or imagingagents. The recombinant polypeptides of the invention, and multimersthereof, have broad application for delivery of any diagnostic, imagingagent, or other compound to epithelial tissue comprising intercellularjunctions where access to a target of interest can be limited. Invarious non-limiting embodiments, the imaging agents can include anychemical compound that can produce a detectable signal, either directlyor indirectly. Many such imaging agents are known to those of skill inthe art. Examples of imaging agents suitable for use in the disclosedmethods and compositions are radioactive isotopes, fluorescentmolecules, magnetic particles (including nanoparticles), metal particles(including nanoparticles), phosphorescent molecules, enzymes,antibodies, ligands, and combinations thereof, while diagnostic agentsmay comprise a compound that is a diagnostic marker for a particularepithelial disorder bound to the such an imaging agent. Methods fordetecting and measuring signals generated by imaging agents are alsoknown to those of skill in the art. For example, radioactive isotopescan be detected by scintillation counting or direct visualization;fluorescent molecules can be detected with fluorescentspectrophotometers; phosphorescent molecules can be detected with aspectrophotometer or directly visualized with a camera; enzymes can bedetected by detection or visualization of the product of a reactioncatalyzed by the enzyme; antibodies can be detected by detecting asecondary detection label coupled to the antibody. In one preferredembodiment, the imaging agent and/or diagnostic is one that can be usedto detect a tumor, whether by direct tumor binding, or by coupling ofthe imaging or diagnostic agent with a compound that can bind the tumor.

In various embodiments, the imaging agent can be a fluorescent imagingagent, while diagnostic agents may comprise a compound that is adiagnostic marker for a particular epithelial disorder bound to thefluorescent imaging agent. A fluorescent imaging agent is any chemicalmoiety that has a detectable fluorescence signal. This imaging agent canbe used alone or in combination with other imaging agents. Examples ofsuitable fluorescent agents that can be used in the compositions andmethods disclosed herein include, but are not limited to, fluorescein(FITC), 5-carboxyfluorescein-N-hydroxysuccinimide ester,5,6-carboxymethyl fluorescein, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD),fluorescamine, OPA, NDA, indocyanine green dye, the cyanine dyes (e.g.,Cy3, Cy3.5, Cy5, Cy5.5 and Cy7),4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine,acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenylinaphthalimide-3,5disulfonate, N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BODIPY,Brilliant Yellow, coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151), cyanosine,4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), eosin, eosinisothiocyanate, erythrosin B, erythrosine, isothiocyanate, ethidiumbromide, ethidium, 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate, IR144, IR1446, Malachite Green isothiocyanate,4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine,pararosaniline, Phenol Red, B-phycoerythrin, o-phthaldialdehyde, pyrene,pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4(Cibacron[R] Brilliant Red 3B-A), 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), 5,6-tetramethyl rhodamine, rhodamine B, rhodamine 123,rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid,coumarin-6, and the like, including combinations thereof. Thesefluorescent imaging moieties can be obtained from a variety ofcommercial sources, including Molecular Probes, Eugene, Oreg. andResearch Organics, Cleveland, Ohio, or can be synthesized by those ofordinary skill in the art.

In another example, the imaging agents can comprise a Magnetic ResonanceImaging (MRI) agent, while diagnostic agents may comprise a compoundthat is a diagnostic marker for a particular epithelial disorder boundto the MRI agent. A MRI agent is any chemical moiety that has adetectable magnetic resonance signal or that can influence (e.g.,increase or shift) the magnetic resonance signal of another agent. Thistype of imaging agent can be used alone or in combination with otherimaging agent. In still another example, a gadolinium-based MRI agentcan serve as an imaging agent. An example of a suitable MRI agent thatcan be incorporated into the disclosed imaging agents ispara-amino-benzyl diethylenetriaminepentaacetic acid (p-NH₂-Bz-DTPA,Compound 7), a conjugable form of diethylenetriaminepentaacetic acid(DTPA), which is known to strongly bind gadolinium and is approved forclinical use as a magnetic resonance contrast agent. Incorporation of anMRI agent on a large macromolecule such as a dendrimeric substrate asdisclosed herein can allow large Ti relaxation (high contrast) andmultiple copies of agent on a single molecule, which can increasesignal. By combining an MRI imaging agent and, for example, afluorescent imaging agent, the resulting agent can be detected, imaged,and followed in real-time via MR I. Other imaging agents include PETagents that can be prepared by incorporating an 18F or a chelator for64Cu or 68Ga. Also, addition of a radionuclide can be used to facilitateSPECT imaging or delivery of a radiation dose, while diagnostic agentsmay comprise a compound that is a diagnostic marker for a particularepithelial disorder bound to the PET agent.

In some embodiments, the diagnostic agent is a diagnostic imaging agent,including but not limited to position emission tomography (PET) agents,computerized tomography (CT) agents, magnetic resonance imaging (MRI)agents, nuclear magnetic imaging agents (NMI), fluoroscopy agents andultrasound contrast agents. Such diagnostic agents include radioisotopesof such elements as iodine (I), including ¹²³I, ¹²⁵I, ¹³¹I etc., barium(Ba), gadolinium (Gd), technetium (Tc), including ⁹⁹Tc, phosphorus (P),including ³¹P, iron (Fe), manganese (Mn), thallium (Tl), chromium (Cr),including ⁵¹Cr, carbon (C), including ¹⁴C, or the like, fluorescentlylabeled compounds, or their complexes, chelates, adducts and conjugates.Any suitable PET agents can be used, including but not limited tocarbon-11, nitrogen-13, oxygen-15, fluorine-18,11C-metomidate, andglucose analogues thereof, including but not limited to fludeoxyglucose(a glucose analog labeled with fluorine-18.

In other embodiments, the diagnostic agent is a marker gene that encodeproteins that are readily detectable when expressed in a cell(including, but not limited to, beta-galactosidase, green fluorescentprotein, luciferase, and the like) and labeled nucleic acid probes(e.g., radiolabeled or fluorescently labeled probes). In someembodiments, covalent conjugation of diagnostic or imaging agents to theAdB-2/3 multimers provided herein is achieved according to a variety ofconjugation processes. In other embodiments, the diagnostic agent isnon-covalently associated with AdB-2/3 multimers provided.

In another aspect, the present invention provides nucleic acids encodingthe polypeptide or any embodiment of the invention. The nucleic acidsmay comprise RNA or DNA, and can be prepared and isolated using standardmolecular biological techniques, based on the teachings herein. Thenucleic acids may comprise additional domains useful for promotingexpression and/or purification of the encoded protein, including but notlimited to polyA sequences, modified Kozak sequences, and sequencesencoding epitope tags, export signals, and secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In a further aspect, the present invention provides recombinantexpression vectors comprising the nucleic acid of any aspect of theinvention operatively linked to a promoter. “Recombinant expressionvector” includes vectors that operatively link a nucleic acid codingregion or gene to any promoter capable of effecting expression of thegene product. The promoter sequence used to drive expression of thedisclosed nucleic acids in a mammalian system may be constitutive(driven by any of a variety of promoters, including but not limited to,CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number ofinducible promoters including, but not limited to, tetracycline,ecdysone, steroid-responsive). The construction of expression vectorsfor use in transfecting prokaryotic cells is also well known in the art,and thus can be accomplished via standard techniques. (See, for example,Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin,Tex.). The expression vector must be replicable in the host organismseither as an episome or by integration into host chromosomal DNA, andmay comprise any other components as deemed appropriate for a given use,including but not limited to selection markers such as anantibiotic-resistance gene.

In a still further aspect, the present invention provides host cellscomprising the recombinant expression vectors disclosed herein, andprogeny thereof, wherein the host cells can be either prokaryotic oreukaryotic. The cells can be transiently or stably transfected. Suchtransfection of expression vectors into prokaryotic and eukaryotic cellscan be accomplished via any technique known in the art, including butnot limited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc.New York, N.Y.). Techniques utilizing cultured cells transfected withexpression vectors to produce quantities of polypeptides are well knownin the art.

In another aspect, the present invention provides pharmaceuticalcompositions, comprising

(a) an AdB-2/3 fiber multimer of the present invention; and

(b) a pharmaceutically acceptable carrier.

The AdB-2/3 fiber multimer can be any such multimer as described hereinaccording to any aspect, embodiment, or combination of embodiments ofthe invention that incorporates a mutant knob domain polypeptide of anyembodiment of the first aspect of the invention (i.e.: SEQ ID NOS:1-11).

The pharmaceutical composition may further comprise one or moretherapeutic for treating a disorder associated with epithelial tissue,including but not limited to those disclosed above. In a preferredembodiment, the therapeutic is an anti-tumor therapeutic and comprises achemotherapeutic or anti-tumor monoclonal antibody as described herein.In a further preferred embodiment, the anti-tumor therapeutic comprisesan antibody selected from the group consisting of trastuzumab,cetumiximab, petuzumab, Apomab, conatumumab, lexatumumab, bevacizumab,bevacizumab, denosumab, zanolimumab, lintuzumab, edrecolomab, rituximab,ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab,gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab,catumaxomab, ofatumumab, and ibritumomab.

The pharmaceutically acceptable carrier is non-toxic, biocompatible andis selected so as not to detrimentally affect the biological activity ofthe multimers (and any other therapeutic agents combined therewith).Exemplary pharmaceutically acceptable carriers for peptides aredescribed in U.S. Pat. No. 5,211,657 to Yamada. The compositions may beformulated into preparations in solid, semi-solid, gel, liquid orgaseous forms such as tablets, capsules, powders, granules, ointments,solutions, suppositories, inhalants, and injections, allowing for oral,parenteral, or surgical administration. Suitable carriers for parenteraldelivery via injectable, infusion, or irrigation and topical deliveryinclude distilled water, physiological phosphate-buffered saline, normalor lactated Ringer's solutions, dextrose solution, Hank's solution, orpropanediol. In addition, sterile, fixed oils may be employed as asolvent or suspending medium. For this purpose any biocompatible oil maybe employed including synthetic mono- or diglycerides. In addition,fatty acids, such as oleic acid, find use in the preparation ofinjectables. The carrier and agent may be compounded as a liquid,suspension, polymerizable or non-polymerizable gel, paste or salve. Thecarrier may also comprise a delivery vehicle to sustain (i.e., extend,delay, or regulate) the delivery of the agent(s) or to enhance thedelivery, uptake, stability, or pharmacokinetics of the therapeuticagent(s). Such a delivery vehicle may include, by way of non-limitingexample, microparticles, microspheres, nanospheres, or nanoparticlescomposed of proteins, liposomes, carbohydrates, synthetic organiccompounds, inorganic compounds, polymeric or copolymeric hydrogels, andpolymeric micelles. Suitable hydrogel and micelle delivery systemsinclude the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexesdisclosed in International Publication No. WO 2004/009664 A2, and thePEO and PEO/cyclodextrin complexes disclosed in U.S. Publication No.2002/0019369 A1. Such hydrogels may be injected locally at the site ofintended action, or subcutaneously or intramuscularly to form asustained release depot.

For intrathecal (IT) or intracerebroventricular (ICV) delivery,appropriately sterile delivery systems (e.g., liquids; gels,suspensions, etc.) can be used to administer the compositions. For oraladministration of non-peptidergic agents, the compositions may becarried in an inert filler or diluent such as sucrose, cornstarch, orcellulose.

The compositions of the present invention may also include biocompatibleexcipients, such as dispersing or wetting agents, suspending agents,diluents, buffers, penetration enhancers, emulsifiers, binders,thickeners, flavoring agents (for oral administration). Exemplaryformulations can be parenterally administered as injectable dosages of asolution or suspension of the multimer in a physiologically acceptablediluent with a pharmaceutical carrier that can be a sterile liquid suchas water, oils, saline, glycerol, or ethanol. Additionally, auxiliarysubstances such as wetting or emulsifying agents, surfactants, pHbuffering substances and the like can be present in compositionscomprising modified polypeptides. Additional components ofpharmaceutical compositions include petroleum (such as of animal,vegetable, or synthetic origin), for example, soybean oil and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers for injectable solutions.

The pharmaceutical composition can also be administered in the form of adepot injection or implant preparation that can be formulated in such amanner as to permit a sustained or pulsatile release of the multimersand other therapeutic (if present).

The pharmaceutical composition may comprise in addition to thepolypeptide of the invention (a) a lyoprotectant; (b) a surfactant; (c)a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) apreservative and/or (g) a buffer. In some embodiments, the buffer in thepharmaceutical composition is a Tris buffer, a histidine buffer, aphosphate buffer, a citrate buffer or an acetate buffer. Thepharmaceutical composition may also include a lyoprotectant, e.g.sucrose, sorbitol or trehalose. In certain embodiments, thepharmaceutical composition includes a preservative e.g. benzalkoniumnchloride, benzethonium, chlorohexidine, phenol, nm-cresol, benzylalcohol, methylparaben, propylparaben, chlorobutanol, o-cresol,p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoicacid, and various mixtures thereof. In other embodiments, thepharmaceutical composition includes a bulking agent, like glycine. Inyet other embodiments, the pharmaceutical composition includes asurfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. The pharmaceutical composition mayalso include a tonicity adjusting agent, e.g., a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes a stabilizer, e.g., a molecule which,when combined with a protein of interest substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized or liquid form. Exemplary stabilizers include sucrose,sorbitol, glycine, inositol, sodium chloride, methionine, arginine, andarginine hydrochloride.

The pharmaceutical composition can be packaged in any suitable manner.In one embodiment, the pharmaceutical composition is packaged as a kitcontaining a container (such as a vial) of the AdB-2/3 fiber multimer.In a preferred embodiment, the kit further comprises, in the same or aseparate container (such as a vial), a therapeutic, diagnostic, orimaging agent to be administered to a subject, together with the AdB-2/3fiber multimer.

In a further aspect, the present invention provides kits comprising (a)one or more recombinant polypeptides/AdB-2/3 fiber multimers, isolatednucleic acids, recombinant expression vectors, and/or host cells of theinvention; and (b) instructions for its/their use in treating a disorderassociated with epithelial tissue. The kits may further comprise atherapeutic for use in the methods of the present invention.

In a further aspect, the present invention provides methods forenhancing therapeutic treatment, or diagnosis of a disorder associatedwith epithelial tissue, and/or imaging epithelial tissues, comprisingadministering to a subject in need thereof:

(a) an amount of one or more therapeutics sufficient to treat thedisorder, diagnostic sufficient to diagnose the disorder, and/or imagingagent sufficient to image the epithelial tissue; and

(b) an amount of the AdB-2/3 fiber multimer of the invention, or apharmaceutical composition of the invention, sufficient to enhanceefficacy of the one or more therapeutics, diagnostics, and/or imagingagents.

The methods of this aspect of the invention can be used to enhancingtherapeutic treatment, diagnosis, or imaging of a disorder associatedwith epithelial tissue by improving access for the therapeutic,diagnostic, and/or imaging agent to their target and dissemination inepithelial tissue. While not being bound by any mechanism, the inventorsbelieve this occurs through complementary mechanisms: movement of thetarget receptor from the basolateral to the apical cell surface thusallowing better access to the epithelial tissue target by therapeutics,diagnostics, and/or imaging agents that target the receptor, such asmonoclonal antibodies), and better penetration of the therapeuticthrough disruption of intercellular junctions. DSG2 is the primary highaffinity receptor for AdB-2/3. DSG2 is a calcium-binding transmembraneglycoprotein belonging to the cadherin protein family. In epithelialcells, DSG2 is a component of the cell-cell adhesion structure. Itscytoplasmic tail interacts with a series of proteins that are in directcontact with regulators of cell adhesion and intercellularjunctions/cell morphology. It has been shown that DSG2 is overexpressedin a series of epithelial malignancies including gastric cancer,squamous cell carcinomas, melanoma, metastatic prostate cancer, andbladder cancer.

While not being bound by a specific mechanism of action, the inventorsbelieve that the AdB-2/3 fiber multimer binding to DSG2 serves totrigger transient DSG2-mediated opening of intercellular junctions,which serves to improve access of therapeutics, diagnostics, imagingagents, or any other compound of interest that binds to a target inepithelial cells that would otherwise be trapped to at least some extentin intercellular junctions. Detailed examples of such activity areprovided herein. The methods of the invention can thus be carried outusing any AdB-2/3 fiber multimer of the present invention to triggertransient DSG2-mediated opening of intercellular junctions. Exemplarymultimers comprising one or more AdB-2/3 fiber multimers of theinvention that can be used in these methods include, but are not limitedto, AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles(PtDd) (subviral dodecahedral particles produced by AdB-2/3 during theirreplication), and recombinant AdB-2/3 fiber multimers.

The methods of the invention have broad application for delivery of anytherapeutic, diagnostic, imaging agent, or other compound to epithelialtissue comprising intercellular junctions where access to a target ofinterest can be limited, as DSG2 is widely expressed in epithelialcells. As used herein, a “disorder associated with epithelial tissue” isany disorder wherein therapeutic, diagnostic, or imaging agentadministered to/across epithelial cells/epithelial tissue provides aclinical benefit to a patient, whether in improving therapeutic,diagnostic, and/or imaging efficacy. Such disorders include, but are notlimited to, solid tumors (i.e.: any tumor with epithelial celljunctions), gastrointestinal disorders (including but not limited toirritable bowel syndrome, inflammatory bowel disorder, Crohn's disease,ulcerative colitis, constipation, gastroesophageal reflux disease,Barrett's esophagus, etc.), skin diseases (including but not limited topsoriasis and dermatitis), lung disorders (including but not limited tochronic obstructive pulmonary disease, asthma, bronchitis, pulmonaryemphysema, cystic fibrosis, interstitial lung disease, pneumonia,pancreatic duct disorders, brain disorders (ie: any brain disorder thatcould benefit from improved transport of drugs through the blood-brainbarrier), primary pulmonary hypertension, pulmonary embolism, pulmonarysarcoidosis, tuberculosis, etc.), renal disorders, (including but notlimited to glomerulonephritis), liver diseases (including but notlimited to hepatitis), endocrine disorders (including but not limited todiabetes and thyroid disorders), pancreatic duct disorders (includingbut not limited to pancreatitis), and bile duct disorders (including butnot limited to bile duct obstruction, cholecystitis,choledocholithiasis, gallstones, etc.) and infections of epithelialtissues (including but not limited to cellulitis, pneumonia, hepatitis,and pyelonephritis). In one preferred embodiment, the disorderassociated with epithelial tissue comprises a solid tumor, including butnot limited to breast tumors, lung tumors, colon tumors, rectal tumors,skin tumors, endocrine tumors, stomach tumors, prostate tumors, ovariantumors, uterine tumors, cervical tumors, kidney tumors, melanomas,pancreatic tumors, liver tumors, brain tumors, head and neck tumors,nasopharyngeal tumors, gastric tumors, squamous cell carcinomas,adenocarcinomas, bladder tumors, and esophageal tumors. As will beunderstood by those of skill in the art, such tumors include primarytumors, tumors that are locally invasive, as well as tumors that havemetastasized.

As used herein, “enhancing efficacy” means any increase in therapeutic,diagnostic, and/or imaging efficacy over what would be seen using thetherapeutic, diagnostic, and/or imaging agent alone. For example,measurements of therapeutic efficacy will vary depending on the disorderbeing treated, but are readily identified by an attending physician. Forexample, such increases in efficacy include, but are not limited toincreasing one or more of the following relative to treatment with thetherapeutic alone: (a) reducing the severity of the disorder; (b)limiting or preventing development of symptoms characteristic of thedisorder(s) being treated; (c) inhibiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting orpreventing recurrence of the disorder(s) in patients that havepreviously had the disorder(s); and (e) limiting or preventingrecurrence of symptoms in patients that were previously symptomatic forthe disorder(s). In one non-limiting example, treating a solid tumorprovides an ability to induce egress of tumor receptors from thebasolateral side of epithelial cells to enable improved access andkilling of the tumor.

For cancer, there are standards for defining tumor response and standardmethods of measuring response. These include tumor response, which isdetermined by monitoring the change in tumor size or a serum marker ofdisease. A partial response is more than a 50% reduction in the tumor,while a complete response is defined as complete disappearance of thetumor. Methods used to measure tumors are well known to physicians andinclude physical examination, radiological testing such as CT scans,MRI, PET scans, X-rays as well as serum markers such as prostatespecific antigen, which is used to monitor prostate cancer. Othermeasures of therapeutic efficacy of cancer treatment includemeasurements of time to progression, progression-free survival andoverall survival.

Improved diagnostic efficacy includes any improvement in efficacycompared to administration of the diagnostic alone, including but notlimited to, increasing specificity and/or sensitivity of the diagnostictest. Improved imaging efficacy includes any improvement in efficacycompared to administration of the imaging agent alone, including but notlimited to specificity, sensitivity, reproducibility, contrastenhancement, detection of smaller sites of disease, more accuratedelineation of disease, such as size and shape of diseases, such astumors, abscesses, etc.

In various embodiments, the increase in efficacy is a 5%, 10%, 15%, 20%,25%, 50%, 75%, 100%, or greater benefit compared to efficacy with thetherapeutic, diagnostic, and/or imaging agent alone across a patientpopulation.

Any suitable subject can be treated using the methods of the invention,preferably human subjects.

Any therapeutic, diagnostic, imaging agent, or other compound that cantarget epithelial tissue and whose delivery to epithelial tissue can beimproved by transient opening of intercellular junctions can be used inthe methods of the invention. In one embodiment, the therapeutic isselected from the group consisting of antibodies, immunoconjugates,nanoparticles, nucleic acid therapeutics, and combinations thereof,chemotherapeutics, vaccines, radioactive particle/radiation therapy(“radiation”), cellular immunotherapy including adoptive T-cell therapyand dendritic cell therapy (example: intratumoral penetration ofadministered T-cells), inhaled therapeutics, gene therapy constructs(including but not limited to AdB-2/3 virus as a gene therapy vector,and co-administration with an Ad5-based gene therapy vector), othernucleic acid therapeutics, and combinations thereof.

In various embodiments, the therapeutic is selected from the groupconsisting of alkylating agents, angiogenesis inhibitors, antibodies,antimetabolites, antimitotics, antiproliferatives, aurora kinaseinhibitors, apoptosis promoters (for example, Bcl-xL, Bcl-w and Bfl-1)inhibitors, activators of death receptor pathway, Bcr-Abl kinaseinhibitors, BiTE (Bi-Specific T cell Engager) antibodies, biologicresponse modifiers, cyclin-dependent kinase inhibitors, cell cycleinhibitors, cyclooxygenase-2 inhibitors, growth factor inhibitors, heatshock protein (HSP)-90 inhibitors, demethylating agents, histonedeacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,inhibitors of apoptosis proteins (IAPs) intercalating antibiotics,kinase inhibitors, mammalian target of rapamycin inhibitors, microRNA'smitogen-activated extracellular signal-regulated kinase inhibitors,multivalent binding proteins, non-steroidal anti-inflammatory drugs(NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP)inhibitors, platinum chemotherapeutics, polo-like kinase (Plk)inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs,receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids,small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitorsand the like.

Exemplary therapeutics falling within these various classes include, butare not limited to: docetaxel, doxorubicin, irinotecan, paclitaxel(Taxol®), paclitaxel albumin bound particles (Abraxane®), doxorubicinHCL liposome (Doxil®), BiTE antibodies such as adecatumumab (MicrometMT201), blinatumomab (Micromet MT103) and the like, siRNA-basedtherapeutics, alkylating agents including altretamine, AMD-473, AP-5280,apaziquone, bendamustine, brostallicin, busulfan, carboquone, carmustine(BCNU), chlorambucil, CLORETAZINE.® (laromustine, VNP 40101M),cyclophosphamide, dacarbazine, decitabine, 5′-azacytidine, estramustine,fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU),mafosfamide, melphalan, mitobronitol, mitolactol, nimustine, nitrogenmustard N-oxide, ranimustine, temozolomide, thiotepa, TREANDA®(bendamustine), treosulfan, rofosfamide and the like; angiogenesisinhibitors including endothelial-specific receptor tyrosine kinase(Tie-2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors,insulin growth factor-2 receptor (IGFR-2) inhibitors, matrixmetalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9(MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR)inhibitors, thrombospondin analogs, vascular endothelial growth factorreceptor tyrosine kinase (VEGFR) inhibitors and the like;antimetabolites including ALIMTA® (pemetrexed disodium, LY231514, MTA),5-azacitidine, XELODA® (capecitabine), carmofur, LEUSTAT® (cladribine),clofarabine, cytarabine, cytarabine ocfosfate, cytosine arabinoside,decitabine, deferoxamine, doxifluridine, eflornithine, EICAR(5-ethynyl-1-.beta.-D-ribofuranosylimidazole-4-carboxamide),enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or incombination with leucovorin, GEMZAR® (gemcitabine), hydroxyurea,ALKERAN® (melphalan), mercaptopurine, 6-mercaptopurine riboside,methotrexate, methotrexate analogs (such as trimetrexate andpralatraxate), mycophenolic acid, nelarabine, nolatrexed, ocfosfate,pelitrexol, pentostatin, raltitrexed, Ribavirin, triapine, trimetrexate,S-1, tiazofurin, tegafur, TS-1, vidarabine, and the like; Bcl-2 proteininhibitors including AT-101 ((−)gossypol), GENASENSE® (G3139 oroblimersen (Bcl-2-targeting antisense oligonucleotide)), IPI-194,IPI-565, N-(4-(4-((4′-chloro(1,1′-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobe-nzenesulfonamide)(ABT-737),N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide(ABT-263), GX-070 (obatoclax) and the like; Bcr-Abl kinase inhibitorsinclude DASATINIB® (BMS-354825), GLEEVEC® (imatinib) and the like; CDKinhibitors including AZD-5438, BMI-1040, BMS-032, BMS-387, CVT-2584,flavopyridol, GPC-286199, MCS-5A, PD0332991, PHA-690509, seliciclib(CYC-202, R-roscovitine), ZK-304709 and the like; EGFR inhibitorsincluding ABX-EGF, anti-EGFR immunoliposomes, EGF-vaccine, EMD-7200,ERBITUX® (cetuximab), HR3, IgA antibodies, IRESSA® (gefitinib), TARCEVA®(erlotinib or OSI-774), TP-38, EGFR fusion protein, TYKERB® (lapatinib)and the like; ErbB2 receptor inhibitors include CP-724-714, CI-1033(canertinib), HERCEPTIN® (trastuzumab), TYKERB® (lapatinib), OMNITARG®(2C4, petuzumab), TAK-165, GW-572016 (ionafarnib), GW-282974, EKB-569,PI-166, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti-HER/2neubispecific antibody, B7.her2IgG3, AS HER2 trifunctional bispecificantibodies, mAb AR-209, mAb 2B-1 and the like; histone deacetylaseinhibitors include romidepsin, LAQ-824, MS-275, trapoxin,suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid and the like;HSP-90 inhibitors including 17-AAG-nab, 17-AAG, CNF-101, CNF-1010,CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, MYCOGRAB® (humanrecombinant antibody to HSP-90), NCS-683664, PU24FC1, PU-3, radicicol,SNX-2112, STA-9090 VER49009 and the like; activators of death receptorpathways including TRAIL, antibodies or other agents that target TRAILor death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab,ETR2-ST01, GDC0145, (lexatumumab), HGS-1029, LBY-135, PRO-1762 andtrastuzumab; platinum chemotherapeutics include cisplatin, ELOXATIN®(oxaliplatin) eptaplatin, lobaplatin, nedaplatin, PARAPLATIN®(carboplatin), satraplatin, picoplatin and the like; VEGFR inhibitorsincluding AVASTIN® (bevacizumab), ABT-869, AEE-788, axitinib (AG-13736),AZD-2171, CP-547,632, IM-862, MACUGEN (pegaptamib), NEXAVAR® (sorafenib,BAY43-9006), pazopanib (GW-786034), vatalanib (PTK-787, ZK-222584),SUTENT® (sunitinib, SU-11248), VEGF trap, ZACTIMAThi (vandetanib,ZD-6474) and the like; dendritic cell therapy (sipuleucel-T, Provenge®);topoisomerase inhibitors including aclarubicin, 9-aminocamptothecin,amonafide, amsacrine, becatecarin, belotecan, BN-80915, CAMPTOSAR®(irinotecan hydrochloride), camptothecin, dexrazoxine, diflomotecan,edotecarin, ELLENCE® or PHARMORUBICIN® (epirubicin), etoposide,exatecan, abraxane, irenotecan,10-hydroxycamptothecin, gimatecan,lurtotecan, mitoxantrone, orathecin, pirarbucin, pixantrone, rubitecan,sobuzoxane, SN-38, tafluposide, topotecan and the like; antibodiesincluding AVASTIN® (bevacizumab), CD40-specific antibodies, chTNT-1/B,denosumab, ERBITUX® (cetuximab), HUMAX-CD4® (zanolimumab), IGF IR-specific antibodies, lintuzumab, PANOREX® (edrecolomab), RENCAREX® (WXG250), RITUXAN® (rituximab), ticilimumab, trastuzimab and the like;hormonal therapies including ARIMIDEX® (anastrozole), AROMASIN®(exemestane), arzoxifene, CASODEX® (bicalutamide), CETROTIDE®(cetrorelix), degarelix, deslorelin, DESOPAN® (trilostane),dexamethasone, DROGENIL® (flutamide), EVISTA® (raloxifene), AFEMA®(fadrozole), FARESTON® (toremifene), FASLODEX® (fulvestrant), FEMARA®(letrozole), formestane, glucocorticoids, HECTOROL® (doxercalciferol),RENAGEL® (sevelamer carbonate), lasofoxifene, leuprolide acetate,MEGACE® (megesterol), MIFEPREX® (mifepristone), NILANDRON® (nilutamide),NOLVADEX® (tamoxifen citrate), PLENAXIS® (abarelix), prednisone,PROPECIA® (finasteride), rilostane, SUPREFACT® (buserelin), TRELSTAR®(luteinizing hormone releasing hormone (LHRH)), VANTAS® (Histrelinimplant), VETORYL® (trilostane or modrastane), ZOLADEX® (fosrelin,goserelin) and the like; immunologicals including interferon alpha,interferon alpha-2a, interferon alpha-2b, interferon beta, interferongamma-1a, ACTIMMUNE® (interferon gamma-1b) or interferon gamma-nl,combinations thereof and the like. Other agents include ALFAFERONE®(IFN-alpha), BAM-002 (oxidized glutathione), BEROMUN® (tasonermin),BEXXAR® (tositumomab), CAMPATH® (alemtuzumab), CTLA4 (cytotoxiclymphocyte antigen 4), decarbazine, denileukin, epratuzumab, GRANOCYTE®(lenograstim), lentinan, leukocyte alpha interferon, imiquimod, MDX-010(anti-CTLA-4), melanoma vaccine, mitumomab, molgramostim, MYLOTARG™(gemtuzumab ozogamicin), NEUPOGEN® (filgrastim), OncoVAC-CL, OVAREX®(oregovomab), pemtumomab (Y-muHMFG1), PROVENGE® (sipuleucel-T),sargaramostim, sizofilan, teceleukin, THERACYS® (BacillusCalmette-Guerin), ubenimex, VIRULIZIN® (immunotherapeutic, LorusPharmaceuticals), Z-100 (Specific Substance of Maruyama (SSM)), WF-10(Tetrachlorodecaoxide (TCDO)), PROLEUKIN® (aldesleukin), ZADAXIN®(thymalfasin), ZENAPAX® (daclizumab), ZEVALIN®. (90Y-Ibritumomabtiuxetan) and the like; ofatumumab; biological response modifiers agentsincluding krestin, lentinan, sizofuran, picibanil PF-3512676 (CpG-8954),ubenimex and the like; pyrimidine analogs include cytarabine (ara C orArabinoside C), cytosine arabinoside, doxifluridine, FLUDARA®(fludarabine), 5-FU (5-fluorouracil), floxuridine, GEMZAR®(gemcitabine), TOMUDEX® (ratitrexed), TROXATYL® (triacetyluridinetroxacitabine) and the like; purine analogs including LANVIS®(thioguanine) and PURI-NETHOL® (mercaptopurine); antimitotic agentsincluding batabulin, epothilone D (KOS-862),N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,ixabepilone (BMS 247550), paclitaxel, TAXOTERE® (docetaxel), PNU100940(109881), patupilone, XRP-9881 (larotaxel), vinflunine, ZK-EPO(synthetic epothilone) and the like; and other chemotherapeutic agentssuch as ABRAXANE® (ABI-007), ABT-100 (farnesyl transferase inhibitor),ADVEXIN® (Ad5CMV-p53 vaccine), ALTOCOR® or MEVACOR® (lovastatin),AMPLIGE®. (poly I:poly C12U, a synthetic RNA), APTOSYN® (exisulind),AREDIA® (pamidronic acid), arglabin, L-asparaginase, atamestane(1-methyl-3,17-dione-androsta-1,4-diene), AVAGE® (tazarotene), AVE-8062(combreastatin derivative) BEC2 (mitumomab), cachectin or cachexin(tumor necrosis factor), canvaxin (vaccine), CEAVAC® (cancer vaccine),CELEUK® (celmoleukin), CEPLENE® (histamine dihydrochloride), CERVARIX®(human papillomavirus vaccine), CHOP® (C: CYTOXAN® (cyclophosphamide);H: ADRIAMYCIN® (hydroxydoxorubicin); O: Vincristine (ONCOVIN®); P:prednisone), CYPAT® (cyproterone acetate), combrestatin A4P, DAB(389)EGF(catalytic and translocation domains of diphtheria toxin fused via aHis-Ala linker to human epidermal growth factor) or TransMID-107R®(diphtheria toxins), dacarbazine, dactinomycin,5,6-dimethylxanthenone-4-acetic acid (DMXAA), eniluracil, EVIZON™(squalamine lactate), DIMERICINE® (T4N5 liposome lotion),discodermolide, DX-8951f (exatecan mesylate), enzastaurin, EPO906(epithilone B), GARDASIL® (quadrivalent human papillomavirus (Types 6,11, 16, 18) recombinant vaccine), GASTRIMMUNE®, GENASENSE®, GMK(ganglioside conjugate vaccine), GVAX® (prostate cancer vaccine),halofuginone, histerelin, hydroxycarbamide, ibandronic acid, IGN-101,IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox), IL-13-pseudomonasexotoxin, interferon-.alpha., interferon-.gamma., JUNOVAN® or MEPACT®(mifamurtide), lonafarnib, 5,10-methylenetetrahydrofolate, miltefosine(hexadecylphosphocholine), NEOVASTAT® (AE-941), NEUTREXIN® (trimetrexateglucuronate), NIPENT® (pentostatin), ONCONASE® (a ribonuclease enzyme),ONCOPHAGE® (melanoma vaccine treatment), ONCOVAX® (IL-2 Vaccine),ORATHECIN® (rubitecan), OSIDEM® (antibody-based cell drug), OVAREX® MAb(murine monoclonal antibody), paclitaxel, PANDIMEX® (aglycone saponinsfrom ginseng comprising 20(S)protopanaxadiol (aPPD) and20(S)protopanaxatriol (aPPT)), panitumumab, PANVAC®-VF (investigationalcancer vaccine), pegaspargase, PEG Interferon A, phenoxodiol,procarbazine, rebimastat, REMOVAB® (catumaxomab), REVLIMID®(lenalidomide), RSR13 (efaproxiral), SOMATULINE® LA (lanreotide),SORIATANE® (acitretin), staurosporine (Streptomyces staurospores),talabostat (PT 100), TARGRETIN® (bexarotene), TAXOPREXIN®(DHA-paclitaxel), TELCYTA® (canfosfamide, TLK286), temilifene, TEMODAR®(temozolomide), tesmilifene, thalidomide, THERATOPE® (STn-KLH), thymitaq(2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazolinedihydrochloride), TNFERADE® (adenovector: DNA carrier containing thegene for tumor necrosis factor-.alpha.), TRACLEER® or ZAVESCA®(bosentan), tretinoin (Retin-A), tetrandrine, TRISENOX®. (arsenictrioxide), VIRULIZIN®, ukrain (derivative of alkaloids from the greatercelandine plant), vitaxin (anti-alphavbeta3 antibody), XCYTRIN®(motexafin gadolinium), XINLAY® (atrasentan), XYOTAX® (paclitaxelpoliglumex), YONDELIS® (trabectedin), ZD-6126, ZINECARD® (dexrazoxane),ZOMETA® (zolendronic acid), crizotinib, zorubicin and the like.

In another preferred embodiment, the therapeutic comprises a compoundthat binds to desmoglein-2; preferably a compound that binds to DSG2 andopens up tight junctions.

In other embodiments, the therapeutic comprises radioactiveparticles/radiation therapy. Any suitable radioactive therapy orparticle can be used as deemed appropriate by an attending physician,including but not limited to cobalt-60, iodine-131, iridium-192,strontium-89, samarium 153, rhenium-186 and lead-212.

In a preferred embodiment, the therapeutic is an anti-tumor therapeuticand comprises a chemotherapeutic or anti-tumor monoclonal antibody asdescribed herein. In a further preferred embodiment, the anti-tumortherapeutic comprises an antibody selected from the group consisting oftrastuzumab, cetumiximab, petuzumab, apomab, conatumumab, lexatumumab,bevacizumab, bevacizumab, denosumab, zanolimumab, lintuzumab,edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab,epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomabdaclizumab, panitumumab, catumaxomab, ofatumumab, and ibritumomab.Non-limiting examples of useful anti-tumor mAb and their specific usesare listed in Table 1 above, and as further described in Campoli, M., etal., Principles & Practice of Oncology 23(1&2): 1-19 (2009),incorporated herein by reference.

The monoclonal antibody therapeutics can be any type of monoclonalantibody, including but not limited to standard monoclonal antibodies,humanized monoclonals, fully human antibodies generated from mice orother sources, chimeric monoclonals, and fragments thereof. “Humanizedmonoclonal antibodies” refers to monoclonal antibodies derived from anon-human monoclonal antibody, such as a mouse monoclonal antibody.Alternatively, humanized monoclonal antibodies can be derived fromchimeric antibodies that retain, or substantially retain, theantigen-binding properties of the parental, non-human, monoclonalantibodies but which exhibit diminished immunogenicity as compared tothe parental monoclonal antibody when administered to humans. Forexample, chimeric monoclonal antibodies can comprise human and murineantibody fragments, generally human constant and mouse variable regions.Humanized monoclonal antibodies can be prepared using a variety ofmethods known in the art, including but not limited to (1) graftingcomplementarity determining regions from a non-human monoclonal antibodyonto a human framework and constant region (“humanizing”), and (2)transplanting the non-human monoclonal antibody variable domains, but“cloaking” them with a human-like surface by replacement of surfaceresidues (“veneering”). These methods are disclosed, for example, in,e.g., Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc.Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv.Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536(1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol.31(3):169-217 (1994); and Kettleborough, C. A. et al., Protein Eng.4(7):773-83 (1991). Monoclonal antibodies can be fragmented usingconventional techniques, and the fragments screened for utility in thesame manner as for whole antibodies. For example, F(ab′)₂ fragments canbe generated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments. Fab fragments can be obtained by treating an IgG antibodywith papain; F(ab′) fragments can be obtained with pepsin digestion ofIgG antibody. A F(ab′) fragment also can be produced by binding Fab′described below via a thioether bond or a disulfide bond. A Fab′fragment is an antibody fragment obtained by cutting a disulfide bond ofthe hinge region of the F(ab′)2. A Fab′ fragment can be obtained bytreating a F(ab′)2 fragment with a reducing agent, such asdithiothreitol. Antibody fragment peptides can also be generated byexpression of nucleic acids encoding such peptides in recombinant cells(see, e.g., Evans et al., J. Immunol. Meth. 184: 123-38 (1995)). Forexample, a chimeric gene encoding a portion of a F(ab′)2 fragment caninclude DNA sequences encoding the CH1 domain and hinge region of the Hchain, followed by a translational stop codon to yield such a truncatedantibody fragment molecule. Non-limiting examples of monoclonal antibodyfragments include (i) a Fab fragment, a monovalent fragment consistingessentially of the VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab′)2fragments, bivalent fragments comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consistingessentially of the VH and CH1 domains; (iv) a Fv fragment consistingessentially of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsessentially of a VH domain; and (vi) one or more isolated CDRs or afunctional paratope.

In one preferred embodiment that can be combined with any embodiment orcombination of embodiments of the invention, the disorder comprises aHer-2 positive tumor, and the method comprises co-administering theAdB-2/3 fiber multimer of the invention together with suitablemonoclonal antibody therapy, alone or in combination with achemotherapeutic, radiation, or combinations thereof. In a furtherpreferred embodiment, the monoclonal antibody is trastuzumab. In afurther preferred embodiment that can be combined with any of theseembodiments, the Her-2 positive tumor is selected from the groupconsisting of a breast tumor, a gastric tumor, a colon tumor, and anovarian tumor. In a further preferred embodiment, the method is carriedout on patients who have not responded adequately to trastuzumab, suchas by lack of tumor remission, by tumor relapse, or by development ofresistance to trastuzumab. The methods of these embodiments can also beused to help reduce the dosage of trastuzumab required to obtaintherapeutic efficacy, and can thus serve to limit side effects (such astrastuzumab-associated cardiotoxicity).

In another preferred embodiment that can be combined with any embodimentor combination of embodiments of the invention, the disorder comprisesan EGFR-positive tumor, and the method comprises co-administering theAdB-2/3 fiber multimer together with suitable monoclonal antibodytherapy, alone or in combination with a chemotherapeutic, radiation, orcombinations thereof. In a further preferred embodiment, the monoclonalantibody is cetuximab. In a further preferred embodiment that can becombined with any of these embodiments, the EGFR-positive tumor isselected from the group consisting of a lung tumor, a colon tumor, abreast tumor, a rectal tumor, a head and neck tumor, and a pancreatictumor. In a further preferred embodiment, the method is carried out onpatients who have not responded adequately to cetuximab, such as by lackof tumor remission, by tumor relapse, or by development of resistance tocetuximab. The methods of these embodiments can also be used to helpreduce the dosage of cetuximab required to obtain therapeutic efficacy,and can thus serve to limit side effects (such as acne-like rashes thatoften occur during cetuximab therapy).

In one preferred embodiment that can be combined with any embodiment orcombination of embodiments of the invention, the disorder comprises anepithelial tumor, and the method comprises co-administering the AdB-2/3fiber multimer together with a vascular endothelial growth factor (VEGF)inhibitor, alone or in combination with other chemotherapeutic,radiation, or combinations thereof. Any suitable VEGF inhibitor can beused, including but not limited to bevacizumab.

In a further embodiment that can be combined with any embodiment orcombination of embodiments herein, the methods involving solid tumorsfurther comprise administering a compound capable of degrading tumorstroma proteins. Any suitable compound for degrading tumor stromaproteins can be used, including but not limited to relaxin, collagenase,trypsin, dispase, MMP(metalloproteinase)-1, and MMP8. Delivery of suchcompounds can be by any suitable mechanism, including gene therapy,separate administration with the AdB-2/3 fiber multimer and thetherapeutic, or administration as a conjugate with the AdB-2/3 fiber ortherapeutic.

In a further embodiment that can be combined with any embodiment orcombination of embodiments herein, the methods further compriseadministering the AdB-2/3 multimer in combination with other junctionopeners. As used herein, a “junction opener” is a compound capable oftransiently opening intercellular junctions. Any suitable junctionopeners can be used. In one non-limiting embodiment, the junction openercomprises Zona occludens toxin (Zot), a Vibrio cholerae (V.cholerae)-produced toxin that possess the ability to reversibly alterintestinal epithelial junctions, allowing the passage of macromoleculesthrough mucosal barriers (Fasano et al. (1991) Proc Natl Acad Sci USA88: 5242-5246)]. A Zot-derived hexapeptide (AT-1001) has been developed.In another embodiment, Clostridium perfringens enterotoxin removesclaudins-3 and -4 from the tight junctions to facilitate bacterialinvasion (Sonoda N, et al. (1999) J Cell Biol 147: 195-204.]. In afurther embodiment, oncoproteins encoded by human Ad, HPV, HTLV-1 cantransiently open epithelial junctions by mislocalizing the junctionprotein ZO-1 (Latorre I J, et al. (2005) J Cell Sci 118: 4283-4293). Inother embodiments, several human viruses engage tight junction or othercell junction molecules to achieve entry into epithelial cells. Amongthese viruses are hepatitis C virus (Evans M J, et al. (2007) Nature446: 801-805), reovirus (Barton E S, et al. (2001) Cell 104: 441-451),and herpes simplex virus (Geraghty R J, et al. (1998) Science 280:1618-1620).

In another embodiment, the therapeutic is an inhaled therapeutic. Anysuitable inhaled therapeutic can be used in the methods of theinvention. In various non-limiting embodiments, the inhaled therapeuticis selected from the group consisting of corticosteroids,bronchodilators, beta agonists, anticholinergics, albuterol (PROVENTIL®;VENOLIN®; ACCUNEB®; PROAIR®), levalbuterol (XOPENEX®), pirbutrol(MAXAIR®), ipratropium bromide (ATROVENT®), beclomethasone, budesonide,flunisolide (AEROBID®), fluticasone, triamcinolone acetonide,fluticasone (a corticosteroid) and salmeterol (ADVAIR®), formotorol (along-acting, beta-agonist bronchodilator) and budesonide (acorticosteroid) (SYMICORT®), albuterol (a beta agonist) and ipratropium(COMBIVENT®; an anticholinergic) (budesonide (PULMICORT RESPULES®), andtiopropium (SPIRIVA®; an anticholinergic bronchodilator).

In another embodiment, the compound comprises a diagnostic or imagingagent. The methods of the invention have broad application for deliveryof any diagnostic, imaging agent, or other compound to epithelial tissuecomprising intercellular junctions where access to a target of interestcan be limited. In various non-limiting embodiments, the imaging agentscan include any chemical compound that can produce a detectable signal,either directly or indirectly. Many such imaging agents are known tothose of skill in the art. Examples of imaging agents suitable for usein the disclosed methods and compositions are radioactive isotopes,fluorescent molecules, magnetic particles (including nanoparticles),metal particles (including nanoparticles), phosphorescent molecules,enzymes, antibodies, ligands, and combinations thereof, while diagnosticagents may comprise a compound that is a diagnostic marker for aparticular epithelial disorder bound to the such an imaging agent.Methods for detecting and measuring signals generated by imaging agentsare also known to those of skill in the art. For example, radioactiveisotopes can be detected by scintillation counting or directvisualization; fluorescent molecules can be detected with fluorescentspectrophotometers; phosphorescent molecules can be detected with aspectrophotometer or directly visualized with a camera; enzymes can bedetected by detection or visualization of the product of a reactioncatalyzed by the enzyme; antibodies can be detected by detecting asecondary detection label coupled to the antibody. In one preferredembodiment, the imaging agent and/or diagnostic is one that can be usedto detect a tumor, whether by direct tumor binding, or by coupling ofthe imaging or diagnostic agent with a compound that can bind the tumor.

In one example, the imaging agents can comprise a fluorescent imagingagent, while diagnostic agents may comprise a compound that is adiagnostic marker for a particular epithelial disorder bound to thefluorescent imaging agent. A fluorescent imaging agent is any chemicalmoiety that has a detectable fluorescence signal. This imaging agent canbe used alone or in combination with other imaging agents. Examples ofsuitable fluorescent agents that can be used in the compositions andmethods disclosed herein include, but are not limited to, fluorescein(FITC), 5-carboxyfluorescein-N-hydroxysuccinimide ester,5,6-carboxymethyl fluorescein, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD),fluorescamine, OPA, NDA, indocyanine green dye, the cyanine dyes (e.g.,Cy3, Cy3.5, Cy5, Cy5.5 and Cy7),4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine,acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenylinaphthalimide-3,5disulfonate, N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BODIPY,Brilliant Yellow, coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151), cyanosine,4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), eosin, eosinisothiocyanate, erythrosin B, erythrosine, isothiocyanate, ethidiumbromide, ethidium, 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate, IR144, IR1446, Malachite Green isothiocyanate,4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine,pararosaniline, Phenol Red, B-phycoerythrin, o-phthaldialdehyde, pyrene,pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4(Cibacron[R] Brilliant Red 3B-A), 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), 5,6-tetramethyl rhodamine, rhodamine B, rhodamine 123,rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid,coumarin-6, and the like, including combinations thereof. Thesefluorescent imaging moieties can be obtained from a variety ofcommercial sources, including Molecular Probes, Eugene, Oreg. andResearch Organics, Cleveland, Ohio, or can be synthesized by those ofordinary skill in the art.

In another example, the imaging agents can comprise a Magnetic ResonanceImaging (MRI) agent, while diagnostic agents may comprise a compoundthat is a diagnostic marker for a particular epithelial disorder boundto the MRI agent. A MRI agent is any chemical moiety that has adetectable magnetic resonance signal or that can influence (e.g.,increase or shift) the magnetic resonance signal of another agent. Thistype of imaging agent can be used alone or in combination with otherimaging agent. In still another example, a gadolinium-based MRI agentcan serve as an imaging agent. An example of a suitable MRI agent thatcan be incorporated into the disclosed imaging agents ispara-amino-benzyl diethylenetriaminepentaacetic acid (p-NH₂-Bz-DTPA,Compound 7), a conjugable form of diethylenetriaminepentaacetic acid(DTPA), which is known to strongly bind gadolinium and is approved forclinical use as a magnetic resonance contrast agent. Incorporation of anMRI agent on a large macromolecule such as a dendrimeric substrate asdisclosed herein can allow large Ti relaxation (high contrast) andmultiple copies of agent on a single molecule, which can increasesignal. By combining an MRI imaging agent and, for example, afluorescent imaging agent, the resulting agent can be detected, imaged,and followed in real-time via MR I. Other imaging agents include PETagents that can be prepared by incorporating an 18F or a chelator for64Cu or 68Ga. Also, addition of a radionuclide can be used to facilitateSPECT imaging or delivery of a radiation dose, while diagnostic agentsmay comprise a compound that is a diagnostic marker for a particularepithelial disorder bound to the PET agent.

In some embodiments, the diagnostic agent is a diagnostic imaging agent,including but not limited to position emission tomography (PET) agents,computerized tomography (CT) agents, magnetic resonance imaging (MRI)agents, nuclear magnetic imaging agents (NMI), fluoroscopy agents andultrasound contrast agents. Such diagnostic agents include radioisotopesof such elements as iodine (I), including ¹²³I, ¹²⁵I, ¹³¹I etc., barium(Ba), gadolinium (Gd), technetium (Tc), including ⁹⁹Tc, phosphorus (P),including ³¹P, iron (Fe), manganese (Mn), thallium (Tl), chromium (Cr),including ⁵¹Cr, carbon (C), including ¹⁴C, or the like, fluorescentlylabeled compounds, or their complexes, chelates, adducts and conjugates.Any suitable PET agents can be used, including but not limited tocarbon-11, nitrogen-13, oxygen-15, fluorine-18,11C-metomidate, andglucose analogues thereof, including but not limited to fludeoxyglucose(a glucose analog labeled with fluorine-18.

In other embodiments, the diagnostic agent is a marker gene that encodeproteins that are readily detectable when expressed in a cell(including, but not limited to, beta-galactosidase, green fluorescentprotein, luciferase, and the like) and labeled nucleic acid probes(e.g., radiolabeled or fluorescently labeled probes). In someembodiments, covalent conjugation of diagnostics agents to the AdB-2/3multimers provided herein is achieved according to a variety ofconjugation processes. In other embodiments, the diagnostic agent isnon-covalently associated with AdB-2/3 multimers provided

In a further aspect, the present invention provides methods forimproving delivery of a substance to an epithelial tissue, comprisingcontacting the epithelial tissue with (a) one or more compound to bedelivered to the epithelial tissue; and (b) an amount of an AdB-2/3fiber multimer of the invention sufficient to enhance delivery of theone or more compounds to the epithelial tissue. In this aspect, thecompounds may be any suitable compound such as those described in detailabove. In a preferred embodiment, the one or more compounds comprise animaging agent. In a further preferred embodiment the epithelial tissuecomprises a solid tumor, including any of those disclosed in the presentapplication. In various non-limiting embodiments, the solid tumor isselected from the group consisting of breast tumors, lung tumors, colontumors, rectal tumors, stomach tumors, prostate tumors, ovarian tumors,uterine tumors, skin tumors, endocrine tumors, cervical tumors, kidneytumors, melanomas, pancreatic tumors, liver tumors, brain tumors, headand neck tumors, nasopharyngeal tumors, gastric tumors, squamous cellcarcinomas, adenocarcinomas, bladder tumors, and esophageal tumors.Exemplary multimers comprising one or more AdB-2/3 fiber multimers ofthe invention that can be used in these methods include, but are notlimited to, AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedralparticles (PtDd) (subviral dodecahedral particles produced by AdB-2/3during their replication), and recombinant AdB-2/3 fiber multimers.

In a still further aspect, the present invention provides methods forimproving delivery of a substance cell or tissue expressing desmoglein 2(DSG2), comprising contacting the cell or tissue expressing DSG2 with(a) one or more compound to be delivered to the cell or tissue; and (b)an amount of an AdB-2/3 fiber multimer of the invention sufficient toenhance delivery of the one or more compounds to the tissue. Exemplarytissue types expressing DSG2 include, but are not limited to epithelialcells/tissue (such as those disclosed herein), human platelets andgranulocytes. As shown in the examples that follow, DSG2 also acts asreceptor in non-polarized cells. Thus, these methods find applicationnot only in epithelial cells and tissue, but also are relevant, forexample, in AdB-2/3 pathogenesis and the intravascular application ofAdB-2/3 vectors for gene therapy purposes. Exemplary multimerscomprising one or more AdB-2/3 fiber multimers of the invention that canbe used in these methods include, but are not limited to, AdB-2/3virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd)(subviral dodecahedral particles produced by AdB-2/3 during theirreplication), and recombinant AdB-2/3 fiber multimers.

In a still further aspect, the present invention provides methods forinducing an epithelial to mesenchymal transition (EMT) in a tissue,comprising contacting the epithelial tissue with an amount of an AdB-2/3fiber multimer of the invention sufficient to induce EMT. EMT is acellular transdifferentiation program where epithelial cells losecharacteristics such as intercellular junctions and gain properties ofmesenchymal cells. EMT is characterized by increased expression ofmesenchymal markers, increased expression of extracellular matrixcompounds, decreased expression of epithelial markers, altered locationof transcription factors, and activation of kinases, and disassociationof intercellular junctions. Exemplary multimers comprising one or moreAdB-2/3 fiber multimers of the invention that can be used in thesemethods include, but are not limited to, AdB-2/3 virions, AdB-2/3capsids, AdB-2/3 dodecahedral particles (PtDd) (subviral dodecahedralparticles produced by AdB-2/3 during their replication), and recombinantAdB-2/3 fiber multimers.

In all of the aspects and embodiments of the methods of the invention,the therapeutic, diagnostic, and/or imaging agent can be administeredtogether with the AdB-2/3 multimer (such as via the compositions of theinvention disclosed above) or may be administered separately. In oneembodiment, the therapeutic and AdB-2/3 multimer are attached, via anysuitable covalent or non-covalent binding. In one non-limitingembodiment, an AbB-2/3 multimer can attached to a toxin or other drug tokill solid tumor cells.

The AdB-2/3 fiber multimer and/or therapeutic can be administered in anyway deemed suitable by an attending physician, depending on whether alocal or systemic mode of administration is most appropriate for thecondition being treated. As used herein, the terms “systemic delivery”and “systemic administration” are intended to include, but are notlimited to, oral and parenteral routes including intramuscular (IM),subcutaneous, intravenous (IV), intra-arterial, inhalational,sublingual, buccal, topical, transdermal, nasal, rectal, vaginal, andother routes of administration that effectively result in dispersementof the delivered agent to a single or multiple sites of intendedtherapeutic action. Preferred routes of systemic delivery for thepresent compositions include intravenous, intramuscular, subcutaneous,and inhalational. In one preferred embodiment, intravenousadministration is used, such as for treatment of disseminated tumors(and for monoclonal antibody delivery). In another embodiment, oraldelivery may be preferred, for example, for treating gastrointestinal(GI) epithelial disorders. In another embodiment, nasal or aerosoldelivery may be preferred for delivery to the lungs, such as for lungepithelial disorders.

The AdB-2/3 fiber multimer may be introduced in association with anothermolecule, such as a lipid or liposome to protect the polypeptides fromenzymatic degradation. For example, the covalent attachment of polymers,especially polyethylene glycol (PEG), has been used to protect certainproteins from enzymatic hydrolysis in the body and thus prolonghalf-life.

The AdB-2/3 fiber multimer and/or therapeutic may be systemicallyadministered on a periodic basis at intervals determined to maintain adesired level of therapeutic effect. For example, administration byintravenous injection may be once per day, once per week, every two tofour weeks or at less frequent intervals. The dosage regimen will bedetermined by the physician considering various factors that mayinfluence the action of the combination of agents. These factors willinclude the extent of progress of the condition being treated, thepatient's age, sex and weight, and other clinical factors. The dosagefor AdB-2/3 fiber multimer and/or therapeutic will vary as a function ofthe multimer and/or therapeutic being administered, as well as thepresence and nature of any drug delivery vehicle (e.g., a sustainedrelease delivery vehicle). In addition, the dosage quantity may beadjusted to account for variation in the frequency of administration andthe pharmacokinetic behavior of the delivered agent(s). Dosage ranges ofAdB-2/3 fiber multimers will generally range between 0.01 and 250 mg/kg,preferably between 0.1 and 10 mg/kg, and more preferably between 0.10 to0.5 mg/kg. Dosages of approved therapeutics are readily identifiable bymedical practitioners. The therapeutic may also be able to beadministered at a reduced dose due to enhanced penetration intoepithelial tissues, such as cancers.

The AdB-2/3 fiber multimer may be administered to the subject before,simultaneously, or after administration of the therapeutic. In apreferred embodiment, administration of the therapeutic and the AdB-2/3fiber multimer are carried out at the same time. The timing ofadministrations of the therapeutic relative to the AdB-2/3 fibermultimer can be varied to achieve the greatest therapeutic effect.Preferably, the therapeutic is administered at a time to ensure itscontact with the transient opening of the intercellular junction causedby AdB-2/3 fiber multimer binding to DSG2. For example, the therapeuticcan be administered prior to, simultaneously with, after eachadministration of the AdB-2/3 fiber multimer. In other preferredembodiments, the therapeutic can be administered after theadministration of the AdB-2/3 fiber multimer, for example up to 5minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours,18 hours, 24 hours, 30 hours, 36 hours, 40 hours, 42 hours, 48 hours, 54hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, oreven up to 96 hours after the administration of the AdB-2/3 fibermultimer.

In another aspect, the present invention provides methods for treating adisorder associated with epithelial tissue, comprising administering toa subject in need thereof an amount of an AdB-2/3 fiber multimer of theinvention sufficient to treat the disorder. In this embodiment, no othertherapeutic is delivered. In non-limiting embodiments, the monotherapyis used to treat a disorder selected from the group consisting of anAdB-2/3 viral infection, a solid tumor, or a disorder that can betreated using an AdB-2/3-based gene delivery vector. For example, intreating solid tumors, the method comprises improving access of immunesystem cells to the site of the disorder, such as by penetration (suchas intratumoral penetration of pre-existing natural killer cells,T-cells or dendritic cells). The method can also be used to treat any ofthe disorders associated with epithelial cells discussed above that canbenefit from improved access of cells of the immune system to the targetepithelial cells. In one preferred embodiment, the disorder is a solidtumor, and the method comprises improving immune system attack of thetumor. The method can be used with any of the solid tumors discussedabove. All embodiments and combinations of embodiments of the firstaspect of the invention can be used in this second aspect as well,unless the context clearly dictates otherwise. Exemplary multimerscomprising one or more AdB-2/3 fiber multimers of the invention that canbe used in these methods include, but are not limited to, AdB-2/3virions, AdB-2/3 capsids, AdB-2/3 dodecahedral particles (PtDd)(subviral dodecahedral particles produced by AdB-2/3 during theirreplication), and recombinant AdB-2/3 fiber multimers.

In another aspect, the present invention provides method for identifyingcandidate compounds for one or more of treating a disorder associatedwith epithelial tissue, improving delivery of a substance to anepithelial tissue, for improving delivery of a substance tissueexpressing DSG2, inducing an EMT in a tissue, and/or treating an AdB-2/3infection comprising (a) contacting an AdB-2/3 fiber multimer of theinvention to DSG2 under conditions to promote multimer binding to DSG2,wherein the contacting is carried out in the presence of one or moretest compounds; and (b) identifying positive test compounds that competewith the AdB-2/3 fiber multimer for binding to DSG2 compared to control;wherein the positive test compounds are candidate compounds for one ormore of treating a disorder associated with epithelial tissue, improvingdelivery of a substance to an epithelial tissue, for improving deliveryof a substance tissue expressing DSG2, inducing an EMT in a tissue,and/or treating an AdB-2/3 infection.

Positive test compounds that compete with the AdB-2/3 fiber multimer forbinding to DSG2 are candidate compounds for transiently openingintracellular junctions through their interaction with DSG2. Follow-upassays to verify the ability of the compounds to transiently openintracellular junctions through their interaction with DSG2 can becarried out by any suitable methods, including but not limited tostudies disclosed in the examples that follow. Compounds so identifiedfor treating a disorder associated with epithelial tissue, improvingdelivery of a substance to an epithelial tissue, improving delivery of asubstance tissue expressing DSG2, or inducing an EMT in a tissue, can beused as substitutes for the AdB-2/3 multimer in any of the methods ofthe present invention. Furthermore, AdB-2/3 represent important humanpathogens causing respiratory tract infections (some sever) andpharyngoconjunctival fever. Thus compounds that can treat AdB-2/3infection would be useful. As disclosed herein, DSG2 as the primaryhigh-affinity receptor used by AdB-2/3, and thus compounds that candiminish AdB-2/3 binding to DSG2 are candidate compounds for treating orlimiting development of AdB-2/3 infection.

Exemplary multimers comprising one or more AdB-2/3 fiber multimers ofthe invention that can be used in these methods include, but are notlimited to, AdB-2/3 virions, AdB-2/3 capsids, AdB-2/3 dodecahedralparticles (PtDd) (subviral dodecahedral particles produced by AdB-2/3during their replication), and recombinant AdB-2/3 fiber multimers. Anysuitable control can be used, including but not limited to an AdB-2/3multimer binding to DSG2 in the absence of test compounds,

In one embodiment, the DSG comprises recombinant DSG2. In anotherembodiment, the methods use cells expressing DSG2 (endogenously orrecombinantly) on the cell surface.

In one non-limiting embodiment, surface plasmon resonance (SPR) studiesusing sensors containing immobilized recombinant DSG2 can be used toidentify candidate compounds that AdB-2/3 fiber multimer binding toDSG2, combined with DSG2 competition studies. In another embodiment, theidentifying comprises transduction studies, where the ability of testcompounds to diminish binding is detected as a decrease in the abilityof functional AdB-2/3 virions to transduce DSG2-expressing epithelialcells. In another embodiment, DSG2-expressing cell extract iselectrophoretically separated and Western blotted, and labeled AdB-2/3fiber multimer is used to probe the Western blot in the presence of thetest compounds. In a further embodiment, dot-blot assays can be used,such as those described in Wang et al., J. Virology (2007)81:12785-12792; and Wang et al. (2008) 82:10567-10579.

When the test compounds comprise polypeptide sequences, suchpolypeptides may be chemically synthesized or recombinantly expressed.Recombinant expression can be accomplished using standard methods in theart, as disclosed above. Such expression vectors can comprise bacterialor viral expression vectors, and such host cells can be prokaryotic oreukaryotic. Synthetic polypeptides, prepared using the well-knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Na-amino protected Na-t-butyloxycarbonyl) amino acid resinwith standard deprotecting, neutralization, coupling and wash protocols,or standard base-labile Na-amino protected 9-fluorenylmethoxycarbonyl(Fmoc) amino acids Both Fmoc and Boc Na-amino protected amino acids canbe obtained from Sigma, Cambridge Research Biochemical, or otherchemical companies familiar to those skilled in the art. In addition,the polypeptides can be synthesized with other Na-protecting groups thatare familiar to those skilled in this art. Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, such as by using automated synthesizers.

When the test compounds comprise antibodies, such antibodies can bepolyclonal or monoclonal. The antibodies can be humanized, fully human,or murine forms of the antibodies. Such antibodies can be made bywell-known methods, such as described in Harlow and Lane, Antibodies; ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1988).

When the test compounds comprise nucleic acid sequences, such nucleicacids may be chemically synthesized or recombinantly expressed as well.Recombinant expression techniques are well known to those in the art(See, for example, Sambrook, et al., 1989, supra). The nucleic acids maybe DNA or RNA, and may be single stranded or double. Similarly, suchnucleic acids can be chemically or enzymatically synthesized by manualor automated reactions, using standard techniques in the art. Ifsynthesized chemically or by in vitro enzymatic synthesis, the nucleicacid may be purified prior to introduction into the cell. For example,the nucleic acids can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the nucleic acids may be used withno or a minimum of purification to avoid losses due to sampleprocessing.

When the test compounds comprise compounds other than polypeptides,antibodies, or nucleic acids, such compounds can be made by any of thevariety of methods in the art for conducting organic chemical synthesis.

EXAMPLES Structural and Functional Studies on the Interaction ofAdenovirus Fiber Knob Domains and Desmoglein 2

Abstract

Human adenovirus (Ad) serotypes Ad3, Ad7, Ad 1, Ad14, and a recentlyemerged new strain of Ad14 (Ad14p1) use the epithelial junction proteindesmoglein 2 (DSG2) as a receptor for infection. Unlike Ad interactionwith CAR and CD46, structural details for Ad binding to DSG2 are stillelusive. Using an approach based on E. coli expression libraries ofrandom Ad3 and Ad14p1 fiber knob mutants we identified amino acidresidues that, when mutated individually, ablated or reduced Ad knobbinding to DSG2. These residues formed three clusters inside one grooveat the extreme distal end of the fiber knob. The Ad3 fiber knob mutantlibrary was also used to identify variants with increased affinity toDSG2. We found a number of mutations within or near to the EF loop ofthe Ad3 knob that resulted in several orders of magnitude higheraffinities to DSG2 compared with the wild-type Ad3 knob. Crystalstructure analysis of one of the mutants showed that the introducedmutations make the EF loop more flexible, which might facilitate theinteraction with DSG2. Our findings have practical relevance for cancertherapy. We have recently reported that an Ad3 fiber knob containingrecombinant protein (JO-1) is able to trigger opening of junctionsbetween epithelial cancer cells, which in turn, greatly improved theintratumoral penetration and efficacy of therapeutic agents. Here weshow that affinity-enhanced versions of JO-1 are therapeutically morepotent than the parental protein in a series of cancer models.

Introduction

We recently identified DSG2 as the main receptor for a group of speciesB adenoviruses, including adenovirus serotype 3 (Ad3), a serotype whichis widely distributed in the human population (42). We found that theDSG2 interacting domain(s) within Ad3 are formed by several fiber knobs(41). This specific mode of Ad3-fiber knob-DSG2 interaction provides ahigh avidity and is functionally relevant for opening of epithelialjunctions (41, 42). The latter involves clustering of DSG2 andactivation of pathways that are reminiscent of anepithelial-to-mesenchymal transition, including the phosphorylation ofMAP kinase and the downregulation of junction protein expression (6, 40,42). The ability to open epithelial junctions appears to be importantfor Ad3 penetration into, and spread within airway epithelial cells(40-42). In a recent study, we attempted to find the minimal moietywithin the Ad3 capsid that confers efficient binding to DSG2 (41). Wegenerated a small recombinant protein, which contains the Ad3 fiber knoband a domain that allows for the self-dimerization of trimeric Ad3 fiberknobs (JO-1) (41). JO-1 can be readily produced in E. coli and purifiedby affinity chromatography. In polarized epithelial cell cultures JO-1triggered the opening of intercellular junctions while intravenousinjection of JO-1 into mice with epithelial tumors allowed for betterpenetration of anti-cancer drugs (5, 6).

The first goal of the present study was to further delineate structuralfeatures of the Ad3 fiber knob-DSG2 interaction. This includedidentifying the amino acid residues within the Ad3 fiber knob that areinvolved in binding to DSG2 and creating JO-1 mutants with reduced andablated binding to DSG2. The second goal of this study, which hastranslational relevance, was to further improve JO-1 by enhancing itsaffinity to DSG2 thereby increasing its therapeutic effect. This wasdone by identifying mutants with increased binding to DSG2.

Both goals were achieved using an E. coli expression library of Ad3fiber knob mutants. We have identified residues in three differentclusters within the Ad3 fiber knob that are critically involved inbinding to DSG2. All residues are localized within one groove at thedistal end of the fiber knob facing the receptor. We then assessed theeffect of these mutations on the fiber knob's ability to open epithelialjunctions by measuring the transepithelial electrical resistance inpolarized epithelial cells in vitro, and the ability to enhance theefficacy of a chemotherapy drug in mice with epithelial xenografttumors. As expected, when mutations with reduced affinity to DSG2 wereintroduced into JO-1, the resulting proteins were less capable to openepithelial junctions. On the other hand, a number of mutations thatincreased the affinity of JO-1 to DSG2 displayed a stronger activity inopening of epithelial junctions. Overall, these studies indicate acorrelation between the affinity of Ad3-fiber knobs to DSG2 andsubsequent effects on epithelial junctions.

The third goal of this study was to delineate the DSG2 interacting fiberknob residues of another DSG2-targeting Ad serotype; the newly emergedstrain Ad14p1 (44), which is considered more pathogenic/virulent thanthe parental strain (Ad14-deWit) (10, 16, 22). The beta sheetdistribution of Ad14p1 differs from that of Ad3, which could potentiallyresult in differences in the mode of DSG2 binding. We thereforegenerated an E. coli expression library of Ad14p1 fiber knob mutants toidentify the DSG2-interacting residues of Ad14p1.

Materials and Methods

Proteins.

Recombinant human DSG2 protein was from Leinco Technologies, Inc. (St.Louis, Mo.). The Ad3 fiber knob was derived from Ad3 virus, GB strain,obtained from the ATCC. The Ad14p1 fiber knob is derived from Ad14p1virus, strain Portland 2971/2007, provided by the Center for DiseaseControl and Prevention (Atlanta, Ga.) (44). The fiber knobs wereproduced in E. coli with N-terminal 6-His tags, using the pQE30expression vector (Qiagen, Valencia, Calif.) and purified by Ni-NTAagarose chromatography as described elsewhere (43).

Cell Lines.

293, HeLa, and A549 cells were maintained in DMEM supplemented with 10%FBS, 100 U/ml penicillin, 100 μg/ml streptomycin (P/S), 2 mM glutamine(Glu) and 1×MEM non-essential-amino-acid solution (Invitrogen, Carlsbad,Calif.). Colon cancer T84 cells (ATCC CCL-248) were cultured in a 1:1mixture of Ham's F12 medium and DMEM, 10% FBS, Glu and P/S. Ovc316 cellsare Her2/neu positive epithelial tumor cells derived from an ovariancancer biopsy (32). Ovc316 cells were cultured in MEGM (Lonza, Mapleton,Ill.), containing 3 μg/L hEGF, 5 g/L insulin, 5 mg/L hydrocortisone, 26mg/L bovine pituitary extract, 25 mg/L amphotericin B) (Lonza),supplemented with 1% FBS, 100 I.U. penicillin, 100 μg/L streptomycin, 10mg/L ciprofloxacin. MDA-MB-231 cells, a triple-negative breast cancercell line (ATCC-HTB-26) were cultured in Leibovitz's L-15 mediumsupplemented with 10% FBS, 100 I.U. penicillin, 100 μg/L streptomycin.TC1-DSG2 were derived from TC1 cells, a C57Bl/6 lung cancer cell linethat expresses HPV16 E6 and E7 (36). TC1 cells were transduced with aVSVG-pseudotyped lentivirus vector expressing human DSG2 (42). A clonethat expressed human DSG2 at a level seen in human tumors was selectedfor in vivo studies.

Adenoviruses.

Propagation, methyl-³H thymidine labeling, purification and titering ofwild-type Ad3 was performed as described elsewhere (37). Ad3-GFP is awild-type Ad3-based vector containing a CMV-GFP expression cassetteinserted into the E3 region (42). Viral particle (VP) concentrationswere determined spectrophotometrically by measuring the optical densityat 260 nm (OD₂₆₀). Titers of plaque forming units (pfu) were performedusing 293 cells as described elsewhere (29). The VP to pfu ratio was20:1 for all virus preparations.

Ad3 Knob Library.

The coding sequence of the Ad3 knob (aa 108-319) containing the last twoshaft repeats was obtained by PCR from Ad3 DNA using primers P1: 5′ATCACGGATCCGGTGGCGGTTCTGGCGGTGGCTCCGGTGGCGGTTCTAACAAACTTT GCAGTAAACTC 3′(SEQ ID NO: 35) and P2: 5′ CTCAGCTAATTAAGCTTAGTCATCTTCTCTAATATAG GA 3′(SEQ ID NO: 36), and cloned into pQE30 (Qiagen, Valencia, Calif.) forexpression in E. coli. The resulting plasmid was called pQE-Ad3knob.Random mutagenic PCR was performed based on a protocol publishedelsewhere (7, 8). Briefly, 20 fmoles pQE-Ad3knob DNA template, 30 pmoles(each) PCR primers (Pmut1:5′-CCAATTCTATTGCACTTAAGAATAACACTTTATGGACAGGT-3′ (SEQ ID NO: 37) andPmut2: 5′-GTCCAAGCTCAGCTAATTAAGCTTAGTCATCTTC-3′ (SEQ ID NO: 38), 2.5 μl,3.5 μl, 5 μl or 10 μl of 10× mutagenic buffer (70 mM MgCl₂, 500 mM KCl,100 mM Tris (pH8.3 at 25° C.), and 0.1% (w/v) gelatin), 10 μl 5 mMMnCl₂, 10 p1 dNTP mix (2 mM dGTP, 2 mM dATP, 10 mM dCTP, 10 mM dTTP) and5 units of Taq polymerase (Promega, Madison, Wis.) were mixed in a finalvolume of 100 μl. PCR conditions were 94° C. for 1 min, 45° C. for 1min, and 72° C. for 1 min (30 cycles). The mutant PCR products (615 bpin length containing mutations only in the reading frame of fiber knobhead) were purified, digested with appropriate enzymes, and cloned intothe plasmid pQE-Ad3knob. For quality control of the random mutageniclibrary, the ligation product was transformed into E. coli M15 (Qiagen,Valencia, Calif.), plated on kanamycin and ampicillin plates, and 50colonies were randomly picked for sequencing.

Ad14 library:

The coding sequence of the Ad14p1 knob (aa 108-323) containing the lasttwo shaft repeats was obtained by PCR from Ad14p1 DNA using primers P1:5′ CATCACGGATCCGGTGGCGGTTCTGGCGGTGGCTCCGGTGGCGGTTCTAATAAACTTTGTACCAAATTGGGAGAAGG 3′ (SEQ ID NO: 39) and P2: 5′GCTAATTAAGCTTAGTCGTCTTCTCTGATGTAGTAAAAGG 3′(SEQ ID NO: 40), and clonedinto pQE30 (Qiagen, Valencia, Calif.) for expression in E. coli. Theresulting plasmid was called pQE-Ad14p1knob. Random mutagenic PCR wasperformed by using PCR primers (Pmut1:5′-AACACCCTGTGGACAGGAGTTAACCC-3′(SEQ ID NO: 41) and Pmut2:5′-CTCAGCTAATTAAGCTTAGTCGTC-3′ (SEQ ID NO: 42)). The mutant PCR products(594 bp in length containing mutations only in the reading frame offiber knob head) were purified, digested with appropriate enzymes, andcloned into the plasmid pQE-Ad14p1knob. For quality control of therandom mutagenic library, the ligation product was transformed into E.coli M15 (Qiagen, Valencia, Calif.), plated on kanamycin and ampicillinplates, and 50 colonies were randomly picked for sequencing.

Colony Assays.

The Ad3 or Ad14p1 knob mutant plasmid library were transformed into XL1Blue or M15 E. coli host strains and plated on LB plates withappropriate antibiotics, i.e, Amp or Amp+Kan, respectively. Afterovernight growth, a 0.45 μm Durapore filter membrane (Millipore,Billerica, Mass.) was placed on top of the colonies. The membrane waspeeled off and placed carefully, with the colonies facing upwards, ontwo sheets of 3 MM paper soaked in LB medium supplemented withantibiotics and 1 mM IPTG. Protein expression of the colonies wasinduced for 6 hours at 30° C., after which the filter with the colonieswas placed on top of a nitrocellulose filter and a Whatman 3 MM papersoaked in native lysis buffer {20 mM Tris-Cl (pH8), 300 mM NaCl, 50 mMMgCl₂, 0.1 mg/ml lysozyme, 0.75 mg/ml DNAse I, ½ complete EDTA-freeprotease inhibitor cocktail tablet/10 ml (Roche, Palo Alto, Calif.)}.The “filter sandwich” was incubated at room temperature for 10 min andthen freeze-thawed 4 times for 10 min at −80° C. and 10 min at 30° C.The nitrocellulose membrane was removed from the sandwich and blockedwith 3% BSA in TBST at 4° C. overnight. The blot was then incubated with0.1 ng/ml of recombinant DSG2 protein (Leinco, St. Louis, Mo.) inTBST/BSA, followed by mouse monoclonal anti-DSG2 antibodies (Clone 6D8;SeroTec Ltd., Oxford, UK) and anti-mouse IgG horseradish peroxidaseconjugate. Colonies without DSG2 binding were picked and cultured in 3ml LB medium overnight. Protein expression was induced with 1 mM IPTGfor 5 hours, the bacteria were then pelleted, resuspended in SDS loadingbuffer and freeze/thawed 3 times. After electrophoresis, proteins weretransferred to nitrocellulose and incubated with anti-His antibodies(MCA1396, Sertec) to assess Ad knob trimerzation. To screen for mutantswith stronger binding to DSG2, the Ad3 knob mutant library wastransformed into M15 E. coli host strain. Protein expression of thecolonies was induced for only 20 min at room temperature. The coloniesthat showed the most intense DSG2 binding signal were picked.

Western Blot:

Mini-PROTEAN precast gels (BIO-RAD, Hercules, Calif.) with 4-15%gradient polyacrylamide were used. A total of 1 μg protein mixed with 2×loading buffer (10 mM Tris-HCl, pH6.8, 200 mM DTT, 4% SDS, 20% glycerol,0.2% bromophenol blue) was loaded per lane. Samples were either boiled(B) for 5 min or loaded unboiled (UB). The following running buffer wasused: 25 mM Tris, pH8.3, 0.192 M glycine, 0.1% SDS. Afterelectrophoresis, proteins were transferred to nitrocellulose andincubated with recombinant human DSG2 protein and anti-DSG2 antibodiesas described previously (42). The Western blots were scanned andquantified using the ImageJ 1.32 software (National Institutes ofHealth, Bethesda, Md.). JO-1 band intensity was set as 100%. Foranalysis of MAP kinase activity, polarized T84 cultures were lysed in 20mM Hepes (pH 7.5), 2 mM EGTA, 10% glycerol, 1% TritonX100, 1 mM PMSF,200 μM Na₃VO₄ and protease inhibitors) on ice. After sonication, sampleswere pelleted and protein containing supernatant stored at −80° C. 15 μgof total protein was used for Western blotting with mAb againstphospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204) (Cell Signaling Danvers,Mass.) or mAb against mouse anti-Erk1/2 (Cell Signaling).

Competition Assays.

HeLa cells were detached from culture dishes by incubation with Verseneand washed with PBS. A total of 10⁵ cells per tube were resuspended in50 al of ice-cold adhesion buffer (DMEM supplemented with 2 mM MgCl₂, 1%FBS, and 20 mM HEPES) containing different concentrations of Ad3 fiberknob protein, and incubated on ice for 1 hour. Then, ³H-labeledwild-type Ad3 virus was added in adhesion buffer at a multiplicity ofinfection (MOI) of 8,000 viral particles (vp) per cell to a final volumeof 100 al. After 1 h of incubation on ice, cells were pelleted andwashed twice with 0.5 ml of ice-cold PBS. After the last wash, thesupernatant was removed and the cell-associated radioactivity wasdetermined by a scintillation counter. The number of VP bound per cellwas calculated by using the virion specific radioactivity and the numberof cells.

Surface Plasmon Resonance:

Acquisitions were done on a BIAcore 3000 instrument. HBS-N(GE-Healthcare, Pittsburgh, Pa.) supplemented with 2 mM CaCl₂ was usedas running buffer in all experiments at a flow rate of 5 μl/min.Immobilization on CM4 sensorchip (BIAcore) was performed using DSG2 at10 μg/ml diluted in 10 mM Acetate buffer pH 4.5 injected for 10 minuteson ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide(NHS) activated flow-cell. A control flow-cell was activated by(EDC/NHS) and inactivated by ethanolamine. Different concentrations ofAd3 fiber knob proteins were injected for 3 minutes association followedby 2.5 minutes dissociation time, and the signal was automaticallysubtracted from the background of the ethanolamine deactivated EDC-NHSflow cell. Kinetic and affinity constants were calculated using theBIAeval software.

Crystallography:

Crystallization conditions for wtAd3 and K217E/F224S knob mutants wereusing the service of the High Throughput Screening Lab at HauptmanWoodward Medical Research Institute. For diffraction studies, wtAd3 andK217E/F224S knob mutant were crystallized using the hanging drop method.Crystals were grown using a reservoir solution of 1.65M MgSO₄(7H₂0) inTAPS buffer 0.1M pH9.0 and a protein solution of 15 mg/ml. Crystals werefrozen using a cryoprotectant composed of 85% reservoir and 15% glycerol(v/v). Data collection was performed at 100K on ID14-4 of the ESRF usingthe EDNA pipeline (19). Data were indexed and scaled using XDS/XSCALE(20, 21) and the structure solved by molecular replacement (PDB 1H7Z)with the program PHASER (25). The model was built and refined using COOT(14) and PHENIX (2), respectively (Table 1). The entry “Structure of theadenovirus 3 knob domain K217E and F224S mutant” has been assigned theRCSB ID code rcsb080687 and PDB ID code 4LIY.

TABLE 1 Data collection and refinement statistics. Wavelength (Å) Ad3knob (K217E/F224S mutant) Resolution range (Å) 48.-2.1 (2.175-2.1) Spacegroup P 32 2 1 Unit cell 96.663 96.663 156.399 90 90 120 Totalreflections 222816 (21396) Unique reflections 49784 (4831) Multiplicity4.5 (4.4) Completeness (%) 99.61 (98.96) Mean I/sigma(I) 11.46 (1.89)Wilson B-factor 40.48 R-merge 0.07161 (0.6146) R-meas 0.08092 CC½ 0.998(0.801) CC* 1 (0.943) R-work 0.1759 (0.2670) R-free 0.2012 (0.3133)Number of atoms 4587 macromolecules 4310 ligands 5 water 272 Proteinresidues 553 RMS(bonds) 0.011 RMS(angles) 1.27 Ramachandran 96 favored(%) Ramachandran 0 outliers (%) Clashscore 4.58 Average B-factor 50.00macromolecules 49.80 ligands 126.60 solvent 51.00 Statistics for thehighest-resolution shell are shown in parentheses.

3D Structure:

Pymol software was used to analyze structure. Mutations in the Ad3 knobdomain (pdb 1H7Z) were stained using different colors on the purpleisosurface. Monomers of Ad3 knob mutant K217E/F224S were drawn incolored cartoons with mutations in sticks and overlaid on the graycartoon view of wild type Ad3 fiber knob.

Negative-Stain Electron Microscopy:

Recombinant JO-2 protein was visualized by negative-stain EM to assessits assembly status. The standard mica-carbon preparation was used withprotein at 0.1 mg/ml. Sample was stained using 1% (wt/vol) sodiumsilicotungstate (pH 7.0) and visualized on a JEOL-1200 electronmicroscope at 100 kV.

Permeability Assay.

A total of 5×10⁵ T84 cells were seeded in 12 mm transwell inserts (PETmembrane, with 0.4 μm pore size (Corning, N.Y.) and cultured for >14days until transepithelial electrical resistance (TEER) was stable.Culture medium was changed every 2-3 days. The cells were exposed toDSG2 ligands (20 μg/ml) in adhesion medium (DMEM, 1% FBS, 2 mM MgCl₂, 20mM HEPES) for 15 min at room temperature and TEER was measured andcalculated as described elsewhere (39).

Animal Studies:

All experiments involving animals were conducted in accordance with theinstitutional guidelines set forth by the University of Washington. Micewere housed in specific-pathogen-free facilities. Immunodeficient (CB17)mice [strain name: NOD.CB17-Prkdc^(scid)/J] were obtained from theJackson Laboratory. Human DSG2 transgenic mice contain 90 kb of thehuman DSG2 locus and express hDSG2 at a level and in a pattern similarto humans (40).

A549, MDA-MB-231, and ovc316 xenograft tumors were established byinjection of the corresponding tumor cells into the mammary fat pad (1:1with Matrigel) of CB17 mice. TC1-DSG2 tumors were established bysubcutaneous injection of TC1-DSG2 cells into DSG2 transgenic mice.JO-0, JO-1, JO-2, or JO-4 were intravenously injected one hour beforethe application of chemotherapeutic drugs: Irinotecan/Camptosar™ (PfizerInc., Groton, Conn.), PEGylated liposomal doxorubicin/Lipodox™ (SunPharmaceuticals IN, India), cetuximab/Erbitux™ (ImClone, Somerville,N.J.), nanoparticle albumin conjugated paclitaxel/Abraxane™ (AbraxisBiosciences, Summit, N.J.). Tumor volumes were measured three times aweek. Each treatment group consisted of a minimum of 5 mice. Animalswere sacrificed and the experiment terminated when tumors in one of thegroups reached a volume of 800 mm³ or tumors displayed ulceration.

Anti-JO4 Antibodies:

anti-JO-4 antibody concentrations in human serum samples were measuredby ELISA. Plates will be coated with rabbit polyclonal anti-Ad3 fiberantibodies (42), followed by recombinant JO-4, human serum samples (1:2to 1:1000 dilution), and anti-human IgG-HRP. Serum samples from ovariancancer patients were provided by the Pacific Ovarian Cancer ResearchConsortium.

3D Structure:

Pymol software was used to visualize the 3D structure of the Ad3 fiberknob (MMDB ID: 16945, PDB ID: 1H7Z (13))

Statistical Analysis:

All results are expressed as mean+/−SD. 2-Way ANOVA for multiple testingwas applied. Animal numbers and P values are indicated in the figurelegends.

Results

Residues Critical for DSG2 Binding.

We first focused our work on Ad3. High-affinity binding to DSG2 andsubsequent epithelial junction opening requires several trimeric fiberknobs in a spatial constellation present in the virion, PtDd, ordimerized (trimeric) Ad3 fiber knob (e.g. JO-1) (41). Recombinant(trimeric) fiber knob with two shaft motifs, but without thedimerization domain (“Ad3 knob monomer”) binds to DSG2 with an affinitythat is orders of magnitude less than JO-1, is not able to block Ad3infection, and does not trigger junction opening (37, 41, 42). However,the affinity of “Ad3 knob monomer” is high enough to detect binding inWestern blots in which soluble DSG2 is used as a probe. We thereforeused an E. coli expression library of His-tagged “Ad3 knob monomer”mutants to identify the amino acid residues within the Ad3 fiber knobthat are critical for DSG2 binding. To generate this library we employedmutagenic PCR (7, 8) in a protocol that on average generated one to twoamino acid substitutions per knob. The Ad3 fiber knob library in XL-1blue E. coli was plated on agar plates, knob expression was induced byIPTG, and colonies were screened for DSG2 binding using recombinant DSG2and anti-DSG2 antibodies. A first screening round of ˜10,000 coloniesfor variants that did not bind to DSG2 revealed 240 candidate colonies.When analyzed by Western blot for the 6×His tag, 40 of the 240 coloniesshowed expression of trimeric fiber knob, indicating the absence ofmajor conformational changes. The remaining variants had truncated fiberknobs or did not form trimers. The corresponding 40 plasmids weresequenced. The vast majority of colonies had single amino acidsubstitutions within the fiber knob. If multiple amino acidsubstitutions per knob were encountered, new Ad3 knob genomes containingthe corresponding mutations individually were synthesized. Furtherrounds of colony screening did not uncover other regions, indicatingthat all the DSG2-interacting residues had been found. A total of 8independent mutants were then used for subsequent studies (FIG. 1A). Forall subsequent studies, we generated self-dimerizing forms of the Ad3fiber mutants (FIG. 1B) and purified them by affinity chromatographyusing NTA-Ni columns. The purified dimerized knob mutants were analyzedfor DSG2 binding by Western blot (FIG. 1C-F). All mutants were severelyreduced in binding when compared to JO-1 (i.e. the dimerized formcontaining the wtAd3 fiber knob). Mutants D261N and F265L were almostcompletely ablated for DSG2 binding, while the other mutants haddifferent levels of residual DSG2 binding (3.6%-20% of wt Ad3 knob)(Table 2, “Western blot”). The identified residues, critical for Ad3knob binding to DSG2, were in three different areas of the Ad3 fiberknob within the CD-loop/D-beta sheet (N186D, V189G, S190P), theFG-loop/G-beta sheets (D261N, F265L), and the H-beta sheet/HI-loop(L292A, L296R, E299V) (FIG. 1A). In a 3D model of the Ad3 fiber knob(PDB accession number 1H7Z_A), all identified residues were located atthe apical side of the fiber knob and followed one specific groove inthe knob (FIG. 2). It is noteworthy, that except F265L, all the othersubstitutions resulted in a change of charge in the correspondingresidue. Differences in migrations patterns in polyacrylamide gels (forexample for D261N) indicate that the substitutions have causedconformational changes. We are currently attempting to crystallize thesemutants to analyze their 3D structure.

To create an Ad3 fiber knob, and eventually an Ad3 virus with maximumablation of DSG2 binding we introduced multiple mutations in the threeidentified areas, specifically a combination of N186D and D261N, acombination of D261N and L296R, and a combination of all three N186D,D261N, and L296R. As expected the combination of mutations in all threecritical regions conferred the highest level of ablation (Table 2,“Western Blot), FIG. 1F).

TABLE 2 Analysis of Ad3 fiber mutants. Western blot Inhibition ofInhibition of Residual DSG2 infection in attachment in binding thepresence of the presence of (wtAd3 dimeric knob dimeric knob knob =100%)* (JO-1 = 100%) (JO-1 = 100%) N186D 5.3% 32.7 56.5 V189G 14 54 81S190P 7.1 30.9 73.4 D261N 0 5.3 45 F265L 0 17.6 55.6 L296R 3.6 5.1 73.7E299V 20 50.7 97.3 N186D, D261N 7.0 5.2 23.5 D261, L296R 7.0 1.5 20.1N186D, D261N, 0 0 18.5 L296R The second column shows the quantitativeanalysis of Western blot bands corresponding to Ad3 knob trimers. Theintensity of the wt Ad3 fiber knob was taken as 100%. The data in thethird column reflect the ability of dimeric Ad3 fiber knob mutants toinhibit Ad3-GFP infection (see FIG. 3B). The higher the percentage, thestronger the inhibition. Inhibition by JO-1 (dimeric wtAd3 knob) istaken as 100%. The fourth column shows corresponding data for Ad3 virusattachment. N = 3. Shown are averages. The standard deviation was lessthan 10%.

Because of its relevance as a recently emerged pathogen, we alsogenerated a library of (“monomeric”) Ad14p1 fiber knob mutants. A firstscreening revealed ˜300 candidate colonies for variants that did notbind to DSG2. When analyzed by Western blot for the 6×His tag, 45 of the300 colonies showed expression of trimeric fiber knob. Sequencing ofthese variants revealed 15 independent mutants with reduced binding toDSG2 (FIG. 1A). Interestingly, in spite of a different beta sheetdistribution, the amino-acid residues that were critical for Ad14p1 knobbinding were in the same three regions that were indentified for the Ad3fiber knob. Because of these similarities, we performed the furtherstudies only with selected Ad3 fiber knob mutants.

Functional Validation.

Competition studies were performed on HeLa cells, which express DSG2(42). First we studied the attachment of ³H-labelled Ad3 virus afterpre-incubation of cells with dimeric Ad3 fiber knobs (FIG. 3A).Reduction in Ad3 virus binding was compared to pre-incubation with JO-1,i.e. the dimeric protein that contained the wild-type Ad3 fiber knob.Inhibition of binding by JO-1 was taken as 100%. The mutants L296R,D261N, and F265L blocked Ad3 virus binding the least (5.1, 5.3, and17.6%), followed by mutants S190P, N186D, and E299V (30.9, 32.7, and50.7% reduced binding, respectively) (Table 2, “Attachment”). A similarassay setup was used to measure the ability of dimeric Ad3 knob mutantsto block transduction of HeLa cells by an Ad3-GFP vector. Transductionwas measured based on GFP expression (FIG. 3B). Similar to what weobserved in the attachment study, Ad3-GFP infection was least reduced bypre-incubation with mutants D261N and F265L, followed by mutants N186D,S190P, L296R, and V189G. Taking the DSG2 binding (Western blot),attachment, and infection competition data together, we concluded thatthe area containing residues 261 to 265 is the most critical area inDSG2 binding. The region around residues 186-190 also contributes tobinding, while the region containing residue 299 appears to be onlymarginally involved in binding. Dimeric Ad3 knob mutants with combinedmutations were also analyzed for their ability to compete with Ad3 virusfor attachment (FIG. 3C) and infection (FIG. 3D). The mutant withmutations in all three areas (N185, D261, L296) did not block Ad3binding or infection even at concentrations of 200 ug/ml indicating thatis nearly ablated for DSG2 binding. Notably, when using sCD46 as a probein the Western blot of wild-type Ad3 fibers, no specific binding wasobserved (FIG. 4). This indicates that Ad3 only inefficiently binds toCD46.

Correlation of Reduced DSG2 Binding and Weaker Ability to OpenEpithelial Junctions.

A straightforward assay for the junction opening function of dimeric Ad3knob mutants is based on measuring the transepithelial electricalresistance (TEER) in transwell cell cultures. Epithelial cancer cellsare cultured until the TEER is constant, when major intercellularjunctions are formed. Addition of JO-1 for 1 hour to the apical side ofthe transwell cultures resulted in a rapid decrease in the TEERindicating opening of junctions (FIG. 5A). Incubation with mutant D261Nhad no effect on the TEER. N186D and E299V had intermediate effects thatcorrelated with the residual binding of the corresponding fiber knobs toDSG2. In previous studies, we have also established that JO-1 triggeredchanges in epithelial junctions of xenograft tumors and increased theanti-tumor efficacy of chemotherapeutics with high molecular weights,for example irinotecan. (Irinotecan has a molecular weight of 586.7 Daand is used to treat colon and lung cancer). We used this effect toassess the function of dimeric Ad3 knob mutants in vivo (FIG. 5B).Similarly to what we observed in vitro, JO-1 enhanced irinotecan therapywhile mutants with reduced DSG2 binding had no significant effect onirinotecan efficacy.

Affinity Enhanced Dimeric Ad3 Fiber Knobs.

As outlined above, JO-1 is relevant for cancer therapy. It is thereforeimportant to better understand structural details of its interactionwith DSG2 and create JO-1 mutants with increased affinity to DSG2.Affinity enhancement of biologics is used to: i) decrease theireffective dose, ii) increase their half-lives, iii) potentially increasetheir therapeutic effects, and iv) circumvent the adverse effects ofantibodies generated by patients against the biologic (e.g.neutralization or changes to the pharmokinetics). To make JO-1 analogueswith increased affinity, we screened the E. coli expression library withrandom mutations within JO-1 for variants with increased binding toDSG2. Out of 10,000 colonies plated, twenty colonies with the mostintense DSG2 signals were picked and plasmid DNA was sequenced. Sevendifferent mutants with one or two amino acid substitutions wereidentified: Y250F, K217E+F224S, N293S, V239D, F224L, E248G+K258E, andL277R+N293D. The localization of the residues in the primary and 3Dstructures of the Ad3 fiber knob are shown in FIG. 6. Notably, most ofthe mutations were localized within the EF loop, indicating that thisloop is involved in stabilizing the interaction between Ad3 and DSG2.V239 and Y250 are not exposed at the knob surface suggesting astructural change in the knob rather than an involvement in directbinding to DSG2 (FIG. 6B, right panel). Recombinant mutant dimeric Ad3knob proteins were then purified. To measure the affinity of the mutantsto DSG2, we performed surface plasmon resonance studies. The outcome ofstudies with knobs containing the dimerization domain was complex, mostlikely due to the fact that these mutants formed multimeric complexes.We therefore performed studies with knob proteins lacking thedimerization domain (“no DD”). The association rate constant (k_(a) ork_(on)), and the dissociation rate constant (k_(d) or k_(off)) as wellas the K_(D) of wt Ad3 knob and all knob mutants are shown in FIG. 7. Inagreement with previous studies (41), we found that the wt Ad3 knobwithout the dimerization domain bound to DSG2 only with relatively lowaffinity (K_(D)=10 μM). With the exception of mutantL227R/noDD+N293D/noDD, all mutants identified in the colony blot screenhad higher affinities to DSG2. Notably, the affinities of mutantY250F/noDD or V239D/noDD were 885 or 405-fold higher than that of wtAd3knob/noDD. The high affinity of the different mutants was mainly dueto a faster association to DSG2 rather than a change in the dissociationrate. The only exception to this trend was mutant N293 S/noDD, for whichthe association rate was lowest when compared to other mutants. However,this was partially compensated by a slower dissociation rate. Togetherthese results indicate that wt Ad3 knob (noDD) binding to DSG2 is mostlylimited by a slow association rate that can be improved by a panel ofmutations. These mutations do not appear to modify the stability of thisinteraction but the balance of association versus dissociation,resulting in higher affinities of ligands to the receptor.

To better understand structural elements that enhance binding to DSG2,we performed a more detailed analysis with mutant K217E/F224S.Transmission electron microscopy with uranyl acetate stained K217E/F224Sfiber knobs containing the dimerization domain showed particles with 6knobs representing dimerized trimeric fibers (FIG. 8A, thick arrows andFIG. 8B). Interestingly, under these conditions fiber knobs also formedregularly shaped arrogates (with ˜30 nm diameter) resembling collapsedPtDd (FIG. 8A, thin arrows and FIG. 8C). We then performed x-raycrystallography studies to resolve the structure of the K217E/F224Smutant at the atomic level (FIGS. 8D-H). As expected, the K217E/F224Smutant formed a monotrimer of fiber knobs (FIG. 8E). The 3D structure ofthe mutant was overlaid with that of the wild-type Ad3 fiber knob (FIG.8F-H). This revealed that the EF loop in the K217E/F224S mutant wascompletely disordered. This loop is at the base of the knob domain atthe junction with the fiber shaft. The K217E/F224S mutations maytherefore allow for easier binding by increasing the flexibility of thisloop region.

Correlation of Increased Affinity with Stronger Ability to OpenEpithelial Junctions.

For the following studies we used Ad3 fiber knob forms containing thedimerization domain. To analyze the selected high-affinity mutants, weperformed competition infection studies with Ad3-GFP on HeLa cells andthe dimeric forms of the affinity enhanced Ad3 fiber mutants (FIG. 9A).Based on GFP expression, all dimeric mutants except mutant L277R+N293Dinhibited Ad3-GFP infection significantly more than JO-1. Notably, thenon-dimerized forms of Ad3 fiber knobs with increased affinity to DSG2were unable to act as competitors in transduction studies (FIG. 9B).Higher affinity to DSG2 resulted in an increased capability to openepithelial junctions in transwell cultures (FIG. 9C). Compared to JO-1,the TEER in cultures incubated with the mutants V250F, V239D, andK217E+F224S was significantly higher.

Two of the affinity enhanced version of JO-1, Y293D and V250F wereanalyzed in in vivo assay. We called these mutants JO-2 and JO-4,respectively. The first study was performed in a xenograft model derivedfrom A549 cells similar to the study described for FIG. 5B. FIG. 10Ashows that the affinity-enhanced mutants JO-2 and JO-4 increasedirinotecan therapy significantly more than JO-1 (p<0.05 starting fromday 27). Furthermore, JO-4 (Kd=11.4 nM) was significantly more efficientthan JO-2 (Kd-24.9 nM), indicating a correlation between affinity toDSG2 and therapeutic effect. Additional studies were performed inxenograft tumors derived from ovc316 cells (30, 32). Ovc316 cells areHer2/neu positive epithelial tumor cells derived from an ovarian cancerbiopsy. These cells can undergo EMT and the reverse process,mesenchymal-to-epithelial-transition (MET), under specific conditions invitro and in vivo. A subfraction of ovc316 cells that is positive forNanog, CD133, and E-cadherin is enriched for cancer stem cells, i.e.self-renewing cells with pluripotent potential and tumor forming ability(30). Ovc316 cells therefore closely model the heterogeneity andplasticity seen in tumors in situ. Intravenous injection of JO-1 at adose of 2 mg/kg one hour before injection of PEGylated liposomaldoxorubicin (PLD), a drug that is widely used for chemotherapy ofovarian cancer, significantly increased the treatment efficiency (FIG.10B). Importantly, at a dose of 0.5 mg/kg JO-4 had an even greaterstimulating effect on PLD therapy. Finally, we tested JO-4 in a modelfor triple-negative breast cancer (TNBC). TNBC is characterized by alack or minimal expression of estrogen receptor (ER), progesteronereceptor (PR) and the absence of Her2/neu overexpression. TNBC accountsfor 15% of all breast cancers. Overall survival is poor compared withthat in patients who have other phenotypes. A characteristic feature ofTNBC is high levels of DSG2 and epithelial junctions. Promising clinicalresults in the treatment of TNBS have been achieved with nanoparticle,albumin-conjugated paclitaxel (nab-paclitaxel/Abraxane™) alone or incombination with the EGFR-targeting mAb cetuximab/Erbitux™ (38). Ourstudy showed that JO-4 significantly increased nab-paclitaxel/cetuximabtherapy in a mouse model with orthotopic TNBC tumors (FIG. 10C). Becauseof its therapeutic relevance, we further studied JO-4 in an adequatemouse tumor model. Because Ad3 virus and Ad3 fiber knob derivatives donot bind to mouse cells and tissues we used human transgenic mice thatexpressed human DSG2 in a pattern and at a level similar to humans (40).These mice were subcutaneously implanted with syngeneic TC1-hDSG tumors.When tumors reached a volume of ˜600 mm³, JO-1 or JO-4 was intravenouslyinjected for safety and efficacy studies. Both JO-1 and JO-4 serumconcentrations declined more than one order of magnitude within an hourafter injection, whereby the decline was significantly greater for JO-4(p<0.01 for 1 hr post-injection) (FIG. 11A). After one hourpost-injection JO-1 and JO-4 concentration reached a plateau with ˜100ng/ml. We also analyzed hematological parameters after i.v. JO-1 andJO-4 injection. Blood chemistry did not show abnormal changes. Bloodcell counts were normal except for lymphocyte and platelet number, whichdecreased early after injection (FIG. 11B). Lymphocyte and plateletcounts reached a nadir at 24 hours p.i. with significantly lower numbersfor JO-4 (p<0.01). Interestingly lymphocyte and platelet counts returnedto normal levels faster in JO-4 injected mice than in JO-1 treatedanimals.

JO-1 and JO-4 are virus-derived proteins and immunogenic. Inimmunocompetent mice, serum IgG antibodies against these proteins can bedetected by ELISA two weeks after injection (5). One of theoreticalpremises for affinity enhancement of therapeutic proteins is that itcircumvents neutralizing serum antibodies. To test this, we performedrepeated injections of JO-1 and JO-4 in an immunocompetent hDSG2 mousetumor model with TC1-hDSG2 tumors (FIG. 11C). After two treatment cyclesof JO-1 and PLD, treatment was stopped and tumors were allowed tore-grow. The 3^(rd) and 4^(th) treatment cycles were started on days 28and day 35, respectively. At the time of the 3^(rd) cycle, serumanti-JO-1 antibodies were detectably by ELISA. Importantly, in both, the3^(rd) and 4^(th) treatment cycles JO-1 and JO-4 had an enhancing effecton PLD therapy, whereby the enhancing effect was significantly strongerfor JO-4.

Overall, our functional studies with affinity-enhanced dimeric Ad3 fibermutants demonstrate a correlation between DSG2 affinity and epithelialjunction opening/therapeutic effects.

DISCUSSION

Residues Involved in Ad3 Knob Binding to DSG2.

Unlike Ad interaction with CAR and CD46 (4, 28), structural details onAd interaction with DSG2 interaction are still elusive. Although thecrystal structure of the Ad3 fiber knob has been resolved, for DSG2, the3D structure of only the most distal of the four extracellular domains(ECD) is available (MMDB ID: 59843). However, our previous competitionstudies with monoclonal antibodies against different DSG2 domainsindicated that ECDs 3 and 4 are involved in binding to Ad3 (42). In thisstudy we used mutagenesis-based analyses to identify the amino acidresidues within the Ad3 fiber knob that are critical for binding toDSG2. Mutagenic analysis of DSG2 was not possible because, whenexpressed in E. coli, the protein did not bind to Ad3, indicating thatpost-translational processing is required to create active Ad3 bindingsites within DSG2 (data not shown). The identified residues, criticalfor Ad3 knob binding to DSG2, were in three different areas of the Ad3fiber knob and formed a potential binding pocket localized in a grooveat the distal end of the fiber knob, facing the receptor. Notably,binding of other Ad serotypes to CAR or CD46 primarily involves regionsat the lateral or basal side of the corresponding fiber knobs (27, 43).Our data indicate that Ad3 uses a different binding strategy. We arecurrently performing crystallography studies with dimeric Ad3 fiberknobs and DSG2. Considering that multimeric Ad3 fiber knobs clusterseveral DSG2 molecules (41), it is expected that the 3D structure ofthis complex will be complicated. It remains to be studied whether theresidues critical for Ad3 fiber knob binding to DSG2 will also beinvolved in binding of other species B Ads to DSG2. Notably, while D261,F265, and E299 are conserved in all four DSG2-interacting Ads (Ad3, 7,11, 14), other critical residues (N186, V189, L296) differ between theseserotypes (FIG. 12).

Ad14 is an important research object because of the recent appearance ofa new strain (Ad14p1). Never previously documented in the United States,Ad14p1 was first reported in March and April 2006 during routinesurveillance at several U.S. military-recruit training centers (26).During March-June of the following year, a total of 140 additional casesof confirmed HAdV-B14p1 respiratory illness were reported in patients inOregon, Washington and Texas (3). Thirty eight percent of these patientswere hospitalized, including 17% who were admitted to intensive careunits; 5% of patients died. Outbreaks of HAdV-B14p1 were subsequentlydetected in the other 5 bases and in civilian populations in Washington(1), Oregon (23), Alaska (15), Wisconsin, and Pennsylvania (10, 22) aswell as in Canada (16), China (33) and South Korea (34). At this point,the molecular basis for the high pathogenicity and/or virulence ofAd14p1 is unclear. We attempted to delineate the structural componentsfor Ad14p1 binding to DSG2. The beta sheet distribution of Ad14p1differs from that of Ad3 (FIG. 1A). Therefore, similar toCD46-interacting serotypes (11, 12), it is possible thatDSG2-interacting Ads vary in their binding strategy to DSG2, which couldresult in different DSG2 binding areas. However, the screening of anAd14p1 fiber knob mutant library did not support this hypothesis. Theareas involved in DSG2 binding were essentially the same for Ad3 andAd14p1 fiber knobs. Nevertheless, our finding are relevant for thetreatment of Ad14p1 viremia, specifally for the production of Ad14p1inhibitors or high affinity decoys that can trigger the opsonization ofvirus present in the blood circulation or airway.

It has been reported that, in addition to DSG2, Ad3 can use CD46 as areceptor to infect cells if DSG2 is absent (35). Previously, we foundthat in polarized normal epithelial cells DSG2 is trapped in tightjunctions and not accessible from the apical side, while CD46 is presenton both membrane sides (42). We therefore speculate that CD46 can serveas a relatively inefficient entry receptor for Ad3, while de novoproduced Ad3 and Ad3 penton-dodecahedra interact with DSG2, openepithelial junctions and allow for efficient lateral spread of Ad3 orpenetration into deeper tissue layers and blood circulation. The abilityto individually ablate the Ad3 knob residues that are critical for DSG2and CD46 binding, respectively, should make it possible to prove thishypothesis.

Affinity-Enhanced Fiber Knobs.

Most of the mutations that increased the affinity to DSG2 were localizedwithin the EF loop, indicating that this loop is involved in stabilizingthe interaction between Ad3 and DSG2. Interestingly, unlike Ad7, 11, and14, the Ad3 fiber knob has in this area two additional residues (VL)followed by a proline. This loop could therefore be extended further andthe proline could orient it in a way that might allow for betterinteraction with the receptor. The analysis of the 3D structure of oneof these mutants at the atomic level supports this conclusion. Thesestudies indicate that the introduced mutations make the loop moreflexible, which might facilitate the interaction with DSG2.

The identification of Ad3 knobs with higher affinity than the wt Ad3knob has implications for Ad3-mediated gene therapy. Recently, genetransfer vectors based on Ad3 have shown promise for cancer therapy inclinical trials (18). Theoretically, affinity enhanced Ad3 vectors couldbe used at lower doses and outcompete neutralizing antibodies. Recently,attempts were undertaken to incorporate high affinity ligands intomeasles virus (17) and Ad5-based vectors (3, 9, 45) in order to increaseefficacy and specificity of target cell infection in vivo. Based on ourfindings in this study, a similar strategy can now be pursued for Ad3vectors.

In addition to improving Ad3 vectors, affinity-enhanced versions of JO-1have translational relevance. Most solid tumors are of epithelial originand, although malignant cells are dedifferentiated, they maintainintercellular junctions, a key feature of epithelial cells, both in theprimary tumor as well as in metastatic lesions (5, 31). Theseintercellular junctions represent a protection mechanism against attacksby the host immune system and pose physical barriers that preventintratumoral penetration and dissemination of cancer therapeutics,including monoclonal antibodies and chemotherapy drugs (5, 31). Wheninjected intravenously into mice with xenograft or syngeneic DSG2transgenic tumors, JO-1 markedly enhanced therapeutic effects with avariety of chemotherapy drugs as well as monoclonal antibodies (5, 6).In this study, we have shown that new affinity-enhanced versions of JO-1(e.g. JO-4) increased the efficacy of cancer therapeutics significantlymore than JO-1 (irinotecan, nab-paclitaxel, PEGylated liposomaldoxorubicin, cetuximab) in four tumor models (A549, ovc316, MDA-MB231,and TC1-DSG2). Studies in DSG2 transgenic mice with syngeneic tumorsshowed that serum JO-4 levels rapidly decrease most likely due tobinding to DSG2 on tissues. Previous studies showed that, in addition totumors, lymphocytes and platelets of hDSG2 transgenic express hDSG2(similar to human and monkeys) (40, 42). Along this line we found thatJO-4 injection resulted in a transient reduction of lymphocyte andplatelet counts.

Despite the fact that approximately one third of humans haveneutralizing antibodies against Ad3 (42), in a recent study with serumfrom ovarian cancer patients we found detectable (binding) antibodiesagainst JO-4 in only 10% of patients (N=38) (FIG. 13). However, it iscertain that adaptive immune responses against intravenouslyadministered JO-4 will develop in humans, particularly after repeatedinjection. In this context it is however noteworthy anti-JO-4 antibodiesgenerated after injection into immunocompetent mice appeared not tocritically inhibit the function of JO-4. The data shown in FIG. 11Cdemonstrate that JO-1 and JO-4 continue to be effective after multipletreatment cycles, even in the presence of detectable antibodies.Because, the therapeutic effect after repeated injection wassignificantly greater for JO-4, we speculate that JO-4 is more potentnot only in junction opening, but also in disrupting complexes betweenthe junction opener and serum antibodies.

In summary, our studies uncover important structural details of Ad3 andAd14p1 fiber knob binding to DSG2. It furthermore shows a correlationbetween the affinity of Ad3-fiber knobs to DSG2 and subsequent effectson epithelial junctions. Finally, the generation of affinity-enhancedrecombinant dimeric Ad3 fiber knobs has implications for cancer therapy.

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We claim:
 1. A pharmaceutical composition, comprising: (a) a recombinantAdB-2/3 fiber polypeptide, comprising: (i) one or more AdB-2/3 fiberpolypeptide shaft domain motifs; (ii) an AdB-2/3 fiber polypeptide knobdomain operatively linked to and located C-terminal to the one or moreAdB-2/3 fiber polypeptide shaft domain motifs, wherein the AdB-2/3 fiberpolypeptide knob domain comprises the peptide of any one of SEQ ID NOS5-11; and (iii) one or more non-AdB-2/3-derived dimerization domainsoperatively linked to and located N-terminal to the one or more AdB-2/3fiber polypeptide shaft domain motifs; and (b) a pharmaceuticallyacceptable carrier.
 2. The pharmaceutical composition of claim 1,wherein the AdB-2/3 fiber polypeptide does not include an AdB-2/3 fiberpolypeptide tail domain.
 3. The pharmaceutical composition of claim 1,wherein each shaft domain motif is selected from the group consisting ofan Ad3 fiber polypeptide shaft domain motif, an Ad7 fiber polypeptideshaft domain motif, an Ad11 fiber polypeptide shaft domain motif, an Ad14 fiber polypeptide shaft domain motif, an Ad14a fiber polypeptideshaft domain motif, and combinations thereof.
 4. The pharmaceuticalcomposition of claim 1, wherein the one or more shaft domain motifscomprise 1-22 shaft domain motifs.
 5. The pharmaceutical composition ofclaim 1, wherein each shaft domain motif comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 43-48.
 6. Thepharmaceutical composition of claim 1, wherein the dimerization domaincomprises an amino acid sequence selected from the group consisting ofEVSALEK (SEQ ID NO:24) and/or KVSALKE (SEQ ID NO: 25).
 7. Thepharmaceutical composition of claim 1 wherein the one or more shaftdomain motifs are the shaft domain motif of SEQ ID NO:43.
 8. Thepharmaceutical composition of claim 1, comprising the amino acidsequence selected from the group consisting of (a) (SEQ ID NO: 28)(M/)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENFIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (b) (SEQ ID NO: 29)(M/)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELEYKQTADSSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (c) (SEQ ID NO: 30)(M/-)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLSAGLAPETTQATLITSPFTFSYIREDD; (d) (SEQ ID NO: 31)(M/-)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFDLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (e) (SEQ ID NO: 32)(M/-)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGLVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLEPKYKQTADFSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYEASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; (f) (SEQ ID NO: 33)(M/-)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADLSARGFMPSTTAYPFVLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD; and (g)(SEQ ID NO: 34) (M/-)GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFVLPNAGTHNGNYIFGQCYYEASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD.


9. The pharmaceutical composition of claim 1, wherein the AdB-2/3 fiberpolypeptide contains a single AdB-2/3 fiber polypeptide shaft domainmotif.
 10. The pharmaceutical composition of claim 1, wherein theAdB-2/3 fiber polypeptide comprises a homotrimer of the polypeptide. 11.The pharmaceutical composition of claim 1, wherein the AdB-2/3 fiberhomotrimer of the polypeptide is dimerized.
 12. The pharmaceuticalcomposition of claim 1, further comprising one or more compoundsconjugated to the recombinant AdB-2/3 fiber polypeptide.
 13. Thepharmaceutical composition of claim 12, wherein the one or morecompounds are selected from the group consisting of therapeutics,diagnostics, and imaging agents.
 14. The pharmaceutical composition ofclaim 13, wherein the one or more compounds comprise at least onetherapeutic, wherein the therapeutic is selected from the groupconsisting of antibodies, immunoconjugates, nanoparticles,chemotherapeutics, radioactive particles, viruses, vaccines, cellularimmunotherapy therapeutics, gene therapy constructs, nucleic acidtherapeutics, and combinations thereof.
 15. A-The pharmaceuticalcomposition of claim 1, wherein the pharmaceutically acceptable carrieris saline.
 16. A method for enhancing therapeutic treatment, ordiagnosis of a disorder associated with epithelial tissue, and/orimaging epithelial tissues, comprising administering to a subject inneed thereof: (a) an amount of one or more therapeutics sufficient totreat the disorder, diagnostic sufficient to diagnose the disorder,and/or imaging agent sufficient to image the epithelial tissue; and (b)an amount of the pharmaceutical composition of claim 10, sufficient toenhance efficacy of the one or more therapeutics, diagnostics, and/orimaging agents.
 17. The method of claim 16, wherein the disorderassociated with epithelial tissue is selected from the group consistingof solid tumors, irritable bowel syndrome, inflammatory bowel disorder,Crohn's disease, ulcerative colitis, constipation, gatroesophagealreflux disease, Barrett's esophagus, chronic obstructive pulmonarydisease, asthma, bronchitis, pulmonary emphysema, cystic fibrosis,interstitial lung disease, pneumonia, primary pulmonary hypertension,pulmonary embolism, pulmonary sarcoidosis, tuberculosis, pancreatitis,pancreatic duct disorders, bile duct obstruction, cholecystitis,choledocholithiasis, brain disorders, psoriasis, dermatitis,glomerulonephritis, hepatitis, diabetes, thyroid disorders, cellulitis,infection, pyelonephritis, and gallstones.
 18. A pharmaceuticalcomposition, comprising: a peptide comprising the amino acid sequenceof: GSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCITEYGKONPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKOTADFSARGEMPSTTAYPFSLPNAGTHNENYIFGOCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTOATLITSPETFSYIREDDGSKVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGSGGGSGGGSGGGSNSIALKNNTLWTGPKPEANCIIEYGKQNPDSKLTLILVKNGGIVNGYVTLMGASDYVNTLFKNKNVSINVELYFDATGHILPDSSSLKTDLELKYKQTADFSARGFMPSTTAYPFDLPNAGTHNENYIFGQCYYKASDGALFPLEVTVMLNKRLPDSRTSYVMTFLWSLNAGLAPETTQATLITSPFTFSYIREDD (SEQ ID NO: 31 with the optional N-terminal  methionine absent);

and a pharmaceutically acceptable carrier.
 19. The pharmaceuticalcomposition of claim 18, wherein the pharmaceutically acceptable carrieris saline.